Pyropia haitanensis accounts for approximately 75% of total dried laver production in China. In recent years, advances in cultivation technologies and increasing economic returns, the expansion of cultivation areas has created an urgent demand for high-quality cultivars. The life cycle of P. haitanensis consists of two distinct phases, a filamentous sporophytic stage (conchocelis) and a foliose gametophytic stage, corresponding to indoor seedling propagation and offshore farming, respectively. The primary objective of artificial indoor seedling production is to generate conchospores and propagules for marine cultivation. Therefore, achieving the synchronized development of mature conchosporangial branches and the efficient release of conchospores are critical for ensuring high yield and quality in the subsequent blade stage. Currently, all five nationally certified P. haitanensis cultivars are pure lines established through the transplantation of free-living conchocelis into shells. Free-living conchocelis cultivation enables substrate-independent propagation, reduces dependence on shell substrates and spatial requirements, and minimizes contamination by epiphytic algae. However, these improved cultivars still face challenges related to asynchronous conchocelis maturation and low conchospore release efficiency—commonly referred to as the "difficulty in breeding elite strains" when using free-living conchocelis. Therefore, the key to overcoming this challenge is to precisely regulate the developmental process of free-living conchocelis following transplantation onto shells (seeding), with a particular emphasis on the coordinated control of conchosporangial branch formation and conchospore release. A systematic understanding of the underlying developmental dynamics and molecular regulatory mechanisms is essential for establishing a robust theoretical foundation to enable the accurate manipulation of high-quality conchocelis development. Previous studies identified diacylglycerol kinase (PhDGK1) as a key regulatory gene involved in the maturation of free-living conchocelis. In this study, conchocelis of strain WO84-1 were treated with 1 μmol/L of the diacylglycerol kinase (DGK) inhibitor R59022. Phenotypic differences between the control and treatment groups first became apparent on day 16 of ripening induction, and were evident on day 26. Samples from both groups were collected on day 0, days 16 and 26 for integrated, widely targeted metabolomic profiling and transcriptome sequencing. Metabolomic analysis revealed a significant differential accumulation of key metabolite classes, including amino acids and their derivatives, lipids, flavonoids, alkaloids, terpenoids, and nucleotides and their derivatives. KEGG pathway enrichment analysis indicated that these metabolites were predominantly associated with the biosynthesis of various plant secondary metabolites, pantothenate and CoA biosynthesis, glucosinolate biosynthesis, tyrosine metabolism, efferocytosis, zeatin biosynthesis, and flavonoid biosynthesis. By day 26 of ripening induction, the inhibitor-treated conchocelis exhibited elevated levels of lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and free fatty acids. Except for terpenoids, most amino acid- and nucleotide-related metabolites, along with alkaloids and flavonoids, showed decreasing trends in the treatment group at both time points. Weighted gene co-expression network analysis (WGCNA) clustered transcriptomic data into 15 modules. The turquoise and blue modules contained the largest number of genes (2,111 and 1,981, respectively), whereas the remaining modules contained 71–1,860 genes. Notably, genes within the red module exhibited low expression prior to ripening induction and were highly upregulated in the control group on days 16 and 26 but remained downregulated in the treatment group. KEGG enrichment analysis of genes in the red module revealed significant associations between DNA repair and replication pathways, including homologous recombination, base excision repair, mismatch repair, non-homologous end joining, DNA replication, and nucleotide excision repair. Integrated transcriptome–metabolome correlation analysis identified 22 annotated genes and 72 metabolites. Across both ripening induction stages, the treatment group showed significantly reduced levels of amino acids (and derivatives) and nucleotides (and derivatives), whereas LPC, LPE, and free fatty acids accumulated to substantially higher levels than in the control. DGK activity regulates the balance between phosphatidic acid (PA) and diacylglycerol (DG), thereby influencing the synthesis of LPC and LPE and modulating lipid metabolism. This regulatory function plays a crucial role in maintaining nuclear membrane integrity and ensuring stable expression of genes and transcription factors associated with conchocelis maturation. Through the metabolic intermediate acetyl-CoA, lipid metabolism intersects with amino acid metabolism, alkaloid biosynthesis, flavonoid metabolism, and nucleotide metabolism to form an interconnected metabolic network that coordinately regulates conchocelis development and maturation. Moreover, DGK inhibition disrupts the integrity of the membrane system and exacerbates oxidative stress. Treated conchocelis exhibited activation of multiple stress-related genes, including HSP20 and CAT. HSP20 is involved in abiotic stress responses in Pyropia, upregulation of CAT enhances reactive oxygen species scavenging, and glutathione S-transferase genes contribute to glutathione-mediated redox regulation. Therefore, DGK plays a significant role in conchocelis development and maturation by stabilizing membrane systems, enhancing antioxidant defenses, and maintaining cellular homeostasis under stressful conditions.