In order to study the effect of light color on behavior response, plasma cortisol and blood biochemical indicators of O. mykiss, light stripes of white, red, yellow, blue and green, were respectively laid at the bottom of the tank center at a intensity of 56.9 lx. In each experiment, 10 healthy and lively fish were placed on the same side of the tank to observe the times of O. mykiss crossing the light curtain in the tank. Each experiment lasted for 1 hour. The number of times of fish passing through the light curtain was observed and recorded every 10 minutes, no matter whether it was the first crossing or repeated crossing. Before each experiment, a control group was carried out, that is, a light stripe was laid without turning on the light, and then experiments were carried out in sequence under white light, red light, yellow light, blue light and green light. The experiment was repeated three times for each light color. Blood samples were collected after the fish were exposed to light for 1 hour. Plasma levels of cortisol, total protein (TP), albumin (ALB), triglyceride (TG), total cholesterol (TC), aspartame aminotransferase (AST) and alanine aminotransferase (ALT) under different light colors were determined by enzyme-linked immune reaction kit (ELISA). The results showed as follows: (1) The number of times of O. mykiss crossing the light curtain under yellow and white light conditions was significantly lower than that of the control group and the other three light colors (P<0.05), there was no significant difference between red light, blue light, green light and control group (P>0.05). (2) The plasma cortisol concentration in yellow light condition was significantly higher than that in control group and the other four light colors (P<0.05), there was no significant difference between the control group and the other four light colors (P>0.05). (3) There were significant differences in TP, ALB, TG, TC, AST and ALT among five light colors (P<0.05). This study indicated that the O. mykiss was more adapted to blue light and green light, and had a certain avoidance reaction to yellow and white light, and the effect of yellow light was the strongest, which was consistent with the concentration of cortisol; the O. mykiss adapted to different light color changes through active physiological regulation. In conclusion, green light could be used in O. mykiss production to reduce the stress response. Yellow light stresses fish and is unsuitable for O. mykiss farming, but could be designed as underwater lamp for fishing or preventing fish from escaping from cage. Long-term experiments should be carried out to study the effects of different light colors on behavior, growth, physiological responses of O. mykiss.