To investigate the impact effects of 17a-methyl testosterone on the body length (BL), body weight (BW), body health index (CF), and the anal fin 3rd fin section length (FL), the section number (FJ), and the most wide department width (FW), as well as the level of ARα mRNA and VTGα mRNA expression in female mosquitofish (Gambusia affinis). A total of 250 juvenile female mosquitofish (after birth about 18 d) were exposed to different concentrations of MT (0.5, 5, 50 and 500 nM) in the experimental group, and set the control group and the parallel groups. Exposure experiment lasted for 21 days. The results showed that, the BL and CF in female juvenile exposed to different concentrations of MT for 21 d were not significantly different, only the BW in fish exposed to 50 nM and 500 nM of high-concentration groups was significantly decrease (p<0.05) when compared with the control group; the FJ in 3rd fin of anal fin showed a significant increase, FL was along with extending and FW also showed a significant increase in width (p<0.05); Female juveniles were exposed to different concentrations of MT after 21 d, ARα mRNA expression level was dose-related increase significantly (p<0.05 ), however, VTGα mRNA expression level showed dose-related declinesignificantly (p<0.05 ). The results indicated that the androgenic effects of MT was very obvious, resulting in the morphological masculinization in female mosquitofish during the growth and development process; VTGα and ARα mRNA expression levels could be used as the ideal biomarkers for monitoring the aquaculture water androgen pollutants.