As one of the most essential nutrients in life activities, protein plays a vital role in maintaining the good growth traits and health status of aquatic animals. Rice field culture is the one of the leading aquaculture pattern of Procambarus clarkii in China, which accounts for more than 80% of the annual production. Therefore, we should pay more attention to the protein requirements of P. clarkii farming in the rice field. In order to assess the optimal dietary protein requirement of juvenile P. clarkii farming in the rice field, six isoenergetic and isolipid diets were formulated to contain graded levels of 28%, 31%, 34%, 37%, 40% and 43% crude protein (described as Diet 28-Diet 43 group). Each diet was assigned to a triplicate of 20 experimental crayfish with an average initial body weight of (8.59±0.05) g for eight weeks. At the end of feeding trial, growth performance, muscle composition, hemolymph biochemical indexes, activities of digestive enzyme in intestine, antioxidant enzyme activities and non-specific immunity enzyme activity were measured. Our results showed that: ① The final body weight (FBW), weight gain rate (WGR) and specific growth (SGR) increased at first and then decreased. The meat yield (MY) increased with increasing protein level. ② The dietary protein levels did not significantly affect the muscle moisture, ash and lipid contents. The protein content of the muscle gradually increased as dietary protein level increasing, and then tended to decrease, and was highest in Diet 40 group. ③ The dietary protein level did not significantly affect the content of hemolymph total protein (TP), albumin (ALB), globulin (GLB), glucose (GLU), total cholesterol (TCHO) trended to decrease as dietary lipid level increased. Alanine aminotransferase (ALT) activity and aspartate aminotransferase (AST) activity in hemolymph significantly increased as dietary protein level increasing, both of them in Diet 43 group was highest. ④ The dietary protein level significantly affected the activities of intestine tryptaese and lipase, but not amylase activities. ⑤ Dietary protein also significantly induced the activities of the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), peroxidase (POD), lysozyme (LZM), acid phosphatase (ACP) and total antioxidant capacity (T-AOC), but the contents of malondialdehyde (MDA) was significantly induced. ⑥With dietary protein levels increasing, the expressions of molting-related genes (ecr, chitinase) and genes related immune (hsp70, tlrs, lzm) were firstly significantly increased and then decreased. However, dietary protein level did not significantly affect genes related to protein metabolism (eif4e, s6k1, 4e-bp1). Based on the WGR, broken-line analysis showed that dietary protein requirements of P. clarkii were 40.35% in the present study. In conclusion, under the present experimental conditions and in combination with other factors, the appropriate feed protein level was 40.35%. This study provided a theoretical basis for the development of dedicated feed for P. clarkii farming in the rice field.