Phenylalanine (Phe) is one of the essential amino acids for fish. It is an essential component of proteins that participate in many important physiological and metabolic processes, including growth and development. A deficiency or excess of phenylalanine in the diet can significantly affect the growth performance and/or feed utilization of farmed fish. Therefore, it is necessary to ensure that dietary levels of Phe meet, but do not exceed, the requirements of the fish. The GIFT strain of (Nile tilapia) is becoming an important and economically valuable farmed freshwater species. The aim of this study was to determine the optimal dietary Phe requirement of the GIFT strain by evaluating its growth performance, feed utilization, body composition, muscle amino acid composition, and digestive enzyme activities in a 60-day growth experiment. Triplicate groups of experimental tilapia (initial body weight, 52.70±1.80 g) were cultured in freshwater in 15 floating net cages (1.0 m×1.0 m×1.5 m). Six isonitrogenous (30.10% crude protein) and isocaloric (17.73 MJ/kg gross energy) diets were formulated with protein sources including fish meal, gelatin, peanut meal, soybean meal, casein, and crystalline amino acids. The measured Phe levels in the experimental diets were 0.78%, 0.95%, 1.09%, 1.34%, 1.51%, and 1.72%. The dietary amino acid pattern, except for Phe, was adapted to the muscle amino acid pattern of the GIFT strain. The results showed that with increasing dietary Phe levels, the weight gain rate, specific growth rate, protein efficiency ratio, and protein deposition efficiency increased significantly (<0.05) with the highest values in the 1.34% Phe group. The feed conversion ratio decreased significantly (<0.05) with increasing dietary Phe levels, and showed the lowest value in the 1.09% Phe group. The hepatosomatic index, viscerosomatic index, and condition factor of the GIFT strain all increased significantly (<0.05) as dietary Phe levels increased from 0.78% to 1.34%, but decreased when dietary Phe levels exceeded 1.34%. The highest whole body crude lipid and whole body ash contents were in the 1.34% dietary Phe group. There were no significant differences (>0.05) among the six groups in whole body moisture content, whole body crude protein content, muscle moisture content, muscle crude protein content, muscle crude lipid content, and muscle amino acids contents. The muscle ash content of the group fed 0.98% dietary Phe was significantly (<0.05) lower than that of the group fed 1.72% dietary Phe, but was not significantly different (>0.05) from those of the other groups. The serum chemistry analysis showed that dietary Phe significantly affected the glucose, total cholesterol, triglyceride, and alanine aminotransferase contents (>0.05). The dietary Phe levels also significantly affected the activities of intestinal protease, intestinal lipase, and Na⁺-K⁺-ATPase (<0.05), but not intestinal amylase activity. The quadratic regression analyses of weight gain rate, feed conversion ratio, and protein efficiency ratio showed that the optimum dietary Phe requirement of GIFT is 1.17%-1.21% of the diet or 3.89%-4.02% of dietary proteins.