Vibrio mediterranei is one of the main pathogens caused vibriosis in bivalve including razor clam (Sinonovacula constricta) and Pacific oyster (Crassostrea gigas) in China, resulting in massive mortalities, economic losses, and severe impact on seeding production. In order to construct a quantitative real-time reverse transcription PCR (qRT-PCR) method, Vibrio mediterranei was used as the research object in this study. RNA polymerase alpha-subunit (rpoA), uridylate kinase (pyrH), recombination protein A (recA), L-threonine 3-dehydrogenase (tdh), gyrase B subunit gene (gyrB) and 16S ribosomal RNA gene (16S rRNA) were used as candidate genes to screen reference genes in exponential phase and stationary phase of bacteria. qRT-PCR was used to determine the expression levels of candidate reference genes in V. mediterranei under gradient temperature, salinity, pH, and different medium conditions. The expression stability of candidate genes was comprehensively evaluated by geNorm, NormFinder, BestKeeper, Delta-CT and RefFinder, and the reference genes were screened. The established fluorescence quantitative PCR method was used to analyze the expression of the virulence regulatory gene toxR of V. mediterranei under different environmental conditions. The results showed that the amplification efficiency of the six candidate reference genes was 93.51% -103.58% (R2 > 0.99). The melting curve showed a single peak shape, which met the requirements of qRT-PCR amplification efficiency. tdh was used as an internal reference gene related to temperature changes and different medium stability periods, and rpoA was used as an internal reference gene related to salinity, pH, and exponential phase of different medium. The expression pattern of toxR gene in different culture environments was analyzed by using the established qRT-PCR method. It was found that salinity and temperature influenced the expression of toxR in exponential phase and stationary phase, while temperature had a greater effect on the expression of toxR in exponential phase. The difference in the expression of toxR mainly occurred in the stationary phase of different media. This study evaluated the stability of the internal reference gene of V. mediterranei, which can provide a reference for future research on gene expressions and provide a reference for further research on the expression of pathogenic genes of V. mediterranei.