Poly-β-hydroxybutyrate (PHB) can promote growth and enhance immunity of aquatic animals such as gibel carp, Carassius auratus gibelio. However, the optimal dietary supplementation level and feeding duration for gibel carp are still unclear. In this study, effects of various PHB supplementation levels on gibel carp growth performance, digestive enzyme activities, immune function, disease resistance, and intestinal mucosal barrier were evaluated. Six experimental diets differing in PHB levels were formulated, that is, 0% (P0 as control), 0.5% (P0.5), 1% (P1), 2% (P2), 4% (P4), and 8% (P8). Results indicated that PHB promoted fish growth performance significantly (p < 0.05). Compared to control group P0, the intestinal lipase, protease, and amylase activities in group P4 were significantly increased after 30 days (p < 0.05). Meanwhile, dietary PHB supplementation at 4% level for 30 days and 0.5% for 60 days upregulated the mRNA expression of immune-related