Abstract: Aquatic animals are unable to synthesize n-3 and n-6series of polyunsaturated fatty acids (PUFA) de novo, and mainly obtain PUFA from feed to support growth and reproduction. The proportion of essential fatty acids in diet is crucial to meet these dietary fatty acid requirements. To investigate the ratio of linolenic acid to linoleic acid (ALA/LA) in diet of Procambarus clarkii, six experimental diets were formulated with ALA/LA ratios of 0.14 (R₁), 0.38 (R₂), 0.74 (R₃), 1.28 (R₄), 2.37 (R₅) and 4.54 (R₆). P. clarkii with an initial body weight of (5.99±0.12) g were selected for an 8-week feeding and growth experiment. The results showed that the weight gain rate, specific growth rate, and muscle crude fat content were the highest in the R₃ group, which also had the lowest feed coefficient. The gonad index was the highest in the R₂ group. The R3 group exhibited the highest muscle crude fat and Σn-6 PUFA content, while the R6 group exhibited the highest muscle protein content. The serum glutamic oxalacetic transaminase and glutamic pyruvic transaminase activities were significantly lower in groups with an ALA/LA ratio between 0.38 and 0.74. The dietary ALA/LA ratio significantly affected hepatopancreas and intestinal digestive enzyme activities. The activities of catalase and superoxide dismutase in hepatopancreas reached the maximum in the R₃ and R₄ groups, respectively. The R₃ group had the lowest malondialdehyde content in the hepatopancreas. Additionally, intestinal villus width was significantly increased in the R₃ group, and the number of hepatopancreas B cells was significantly higher in the R₄ group than that in the other groups. The gonadal index of P. clarkii was significantly increased with an ALA/LA ratio between 0.38 and 0.74. The broken-line regression analysis indicated that the optimal dietary ALA/LA ratio for P. clarkii was between 0.45 and 0.46.