This study designed gradient concentration of 0 (control), 3, 6, and 9 g/kg mulberry leaf extract (MLE) added to the essential diet and the initial weight of (2.7 ± 0.2) g healthy Pelophylax nigromaculatus was fed for 56 days to investigate the effects of MLE at different levels on growth performance, carcass composition, and serum biochemical indices. The gradient concentration experiment were labelled as C control group, M03 group, M06 group and M09 group, respectively. Survival rate, final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) initially increased and then decreased with rising MLE levels. The M03 group showed significantly higher FBW, WGR, and SGR by 4.18%, 6.98%, and 2.86%, respectively, compared to the control group(P<0.05). Feed intake rate (FR) and feed conversion ratio (FCR) initially decreased and then increased, with the M03 group exhibiting a significant reduction of 7.73% and 12.32% in FR and FCR (P<0.05). Meanwhile, the M09 group showed no significant difference from the control group (P>0.05). Compared to the control, carcass crude protein and crude lipid in the M03 and M06 groups remained unchanged (P>0.05). The hepatic lipid content significantly decreased by 10.17% and 10.60% (P<0.05). However, carcass protein in the M09 group declined significantly by 4.11% (P<0.05). Serum total cholesterol (TC) and low-density lipoprotein (LDL) in the M03 group decreased significantly by 35.23% and 53.12% (P<0.05). Hepatic superoxide dismutase (SOD) activity in the M03 and M06 groups increased significantly by 18.59% and 9.67% (P<0.05), whereas no significant difference was observed in the M09 group (P>0.05). Hepatic total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels were higher than the control group but not statistically significant (P>0.05). Dietary MLE supplementation improves feed utilization efficiency, antioxidant capacity and growth performance in P. nigromaculatus. Based on a comprehensive evaluation, the optimal MLE inclusion level in the basal diet was recommended to be 0.34%-0.42%. This study provides a theoretical foundation for the green and healthy aquaculture of P. nigromaculatus.