This study was conducted to investigate the effects of phytase in diets containing cottonseed and soybean meal (CS) oil growth performance, feed utilization and digestibility of protein and phosphorus in juvenile olive flounder (initial body weight 2.5 g) Paralichthys olivaceus. Four experimental diets replacing 0%, 30%, 30% and 40% fish meal protein with CS in equal proportion were formulated to be isonitrogenous and isocaloric (designated as CS0, CS30, CS30+P, CS40+P, respectively). Phytase of 1,000 FTU/kg was supplemented in diets CS30+P and CS40+P. Three groups of fish (25 fish per group) were fed one of the experimental diets for 10 weeks. No significant differences were observed in growth performance of fish groups except for the CS40+P diet. Apparent digestibility coefficients of protein and phosphorus in fish fed phytase-containing diets were significantly higher than those of fish fed the CS0 diet. Serum cholesterol was significantly reduced in fish fed the CS-containing diets. Antioxidant activities in the diets and liver of fish were significantly increased with the increment of dietary CS. Gossypol was only detected and found in liver of the fish fed the CS-containing diets, The findings suggest that supplementation of microbial phytase could improve the apparent digestibility of protein and phosphorus in juvenile olive flounder fed the CS-containing diets.