Soybean meal, as a high-quality plant protein source, is widely used in grass carp feed. This study was to estimate the utilization efficiency of grass carp to five non-grain protein sources at different feeding frequencies. A two-factor orthogonal experiment (3×5) was conducted to evaluate the utilization efficiency of soybean meal, Clostridium autoethanogenum protein,Tenebrio molito, chlorella meal and cottonseed protein concentrate for grass carp (Ctenopharyngodon idella) at three feeding frequencies (2, 3 and 4 times/d). Five diets were prepared with five raw materials as a single protein source. Grass carp with an initial body weight of 80 g were fed with the five test diets at three feeding frequencies for 8 weeks in a pond cage (2.0 m×1.5 m×1.0 m), respectively. At the end of culture trial, the growth performance, whole fish body and muscle nutrient composition, and serum biochemical indexes of grass carp were investigated. The results showed that grass carp in Tenebrio molito group (2 times/day) had the lowest growth performance, and the fish in soybean meal (4 times/day) and Clostridium autoethanogenum protein (3 times/day and 4 times/day) groups showed the highest growth performance among all the 15 treatments. Meanwhile, there was significant interaction between protein source and feeding frequency on final weight, feed conversion ratio, protein efficiency ratio and weight gain rate of grass carp (P < 0.05). The protein efficiency of different protein sources (except chlorella meal) for grass carp increased with the increase of feeding frequency. Regardless of feeding frequency, the weight gain rate and protein efficiency rate in chlorella meal and cottonseed concentrate protein groups were significantly lower than those in soybean meal, Clostridium autoethanogenum protein and chlorella meal groups (P < 0.05). The feed conversion ratio showed an opposite trend (P < 0.05). The feeding frequencies and protein sources had no significant effects on body indexes (hepatosomatic index, viscerosomatic index and condition factor) and basic nutrients of whole fish and muscle of grass carp ( P > 0.05). Feeding frequency had significant effects on alkaline phosphatase and glucose in serum of grass carp ( P < 0.05). The alkaline phosphatase activity of groups with 2 times/d was significantly higher than that of groups with 3 times/d and 4 times/d ( P < 0.05). The glucose content of fish fed 4 times/d was significantly higher than that of fed 2 times/d and 3 times/d ( P < 0.05). The activities of alanine aminotransferase and aspartate aminotransferase in serum of grass carps in Tenebrio molito group were significantly higher than those in soybean meal group and chlorella meal group (P < 0.05). Under the present experimental conditions, the optimal feeding frequency of grass carp was 3 times/d or 4 times/d. The order of utilization ability of five protein sources for grass carp was Clostridium autoethanogenum protein = soybean meal = chlorella meal> cottonseed protein concentrate >Tenebrio molitor based on the growth performance.