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A 126‐day experiment was carried out under controlled conditions to compare the effects of five levels (0.5, 1.0, 1.5, 2.0 and 2.5 g kg−1) of dietary nucleotide (Vannagen®) on the growth performance and biological indices of juvenile crayfish (8.25 ± 0.39 g). The protein efficiency ratio (PER, 1.69–2.17), lipid efficiency ratio (LER, 2.29–3.00), energy efficiency ratio (EER, 2.68–3.65), protein productive value (PPV, 57.01–68.95%), lipid productive value (LPV, 63.02–75.15%) and energy productive value (EPV, 68.20–88.15%) increased significantly (P < 0.05) as linear response to increased dietary nucleotide from 0.5 to 2.5 g kg−1 in the feed. With an increase in the dietary nucleotide, the uricase activity (654.29–827.63 U g−1) and lactobacillus count to total count ratio (1.21–2.17) of crayfish fed the experimental diets increased significantly (P < 0.05). Crayfish fed the diets containing different levels of nucleotide (from 0.5 to 2.5 g kg−1) had higher phenoloxidase activity (1.57–2.11 U min−1) than that of control after air exposure challenge. At the levels tested, 2 g kg−1 nucleotide in the diet was considered optimum for growth performance, digestibility and immune responses. It can be concluded that dietary nucleotide exerted positive effects on growth performance, feed utilization and accelerate crayfish immune response against air exposure challenge.

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