The marbled flounder (Pseudopleuronectes yokohamae), characterized by significant female growth superiority, presents a compelling case for all-female aquaculture to address industry constraints in growth performance. Capitalizing on its well-documented XX/XY sex determination system, this study focused on developing an optimized pseudomale (XX♂) induction protocol using 17α-methyltestosterone (MT) to establish monosex female populations for commercial-scale cultivation. Through a 120-day feeding trial with 60-day post hatching (dph) diploid juveniles (initial biometrics: 1.48 ± 0.72 cm body length, 0.07 ± 0.07 g body weight), we systematically evaluated five MT dosage regimens (0-4 mg/kg diet) for their sexual manipulation efficacy and associated biological impacts. Notably, all MT-treated groups achieved complete masculinization (100%), contrasting with the natural 50.0 ± 6.7% male ratio in controls (MT0). Growth metrics at 240 days revealed dose-dependent responses: low-dose groups (MT0.5: 7.61 ± 2.01 cm, 13.14 ± 9.98 g; MT1: 7.27 ± 1.47 cm, 11.40 ± 8.60 g) maintained comparable growth and survival rates (64.0 ± 2.0%) to controls (P > 0.05), while high-dose treatments (MT2/MT4) induced significant growth suppression (P < 0.05). Histological analyses demonstrated preserved testicular microstructure in MT0.5/MT1 groups, featuring distinct spermatogenic lobules and diverse spermatocyte stages, whereas high-dose groups exhibited structural disorganization with diminished spermatocyte populations. Molecular profiling of the meiosis marker sycp3 further validated this dichotomy, showing peak expression in MT1 group and significant downregulation in MT2/MT4 groups relative to controls (P < 0.05), confirming dose-mediated meiotic inhibition. These integrated findings establish 0.5 mg/kg MT as the optimal dosage, successfully reconciling complete sex reversal (100% masculinization) with uncompromised growth performance. This protocol provides a scientifically validated framework for industrial-scale all-female seedling production, effectively addressing both zootechnical and reproductive management challenges in marbled flounder aquaculture.