Abstract: By using Box-Behnken design and response surface methodology, this paper studied the effects of three major environmental factors, i.e., water temperature (20-36 ℃), salinity (0-16), and pH (5.5-8.5), on the sex differentiation of GIFT strain Nile tilapia (Oreochromis niloticus). It was observed that water temperature had highly significant linear and quadratic effects on the sex differentiation, water salinity and pH had non-significant linear and quadratic effects, and the twofactor interactions of the three factors were not significant. The analysis with response surface methodology showed that the male rate of the GIFT tilapia increased with increasing water temperature, and at water temperature 36 ℃, salinity 8, and pH 8.5, the male rate reached the maximum (80%). A model equation about the relationships of the male rate with the three major environmental factors was established， and after eliminating the related non-significant factors, an optimal model equation about the relationship between the male rate and water temperature was attained, which could predict the variation of the GIFT tilapia male rate.