A single factorial experiment was conducted to investigate the effects of Chinese herbal medicines mixture (CHMM) on growth performance, digestive enzyme activity and immune response of Japanese seabass, Lateolabrax japonicus (initial weight 5.01 ± 0.32 g). The fish were fed diets containing six levels of CHMM (0, 4, 8, 12, 16 and 20 g/kg) for 4 weeks. The results showed that the weight gain rate and specific growth rate (SGR) enhanced significantly in fish fed diet containing 8 g/kg CHMM (p < .05), while the feed conversion ratio (FCR) in the 4 and 8 g/kg CHMM groups reduced significantly compared with the control (p < .05). The body crude protein levels in 8 and 16 g/kg groups were significantly higher than the control (p < .05). The pepsin, amylase and lipase activities elevated significantly in the stomach of fish fed 8 g/kg CHMM, while the erepsin and lipase in the intestine of fish in 12 and 16 g/kg groups were higher significantly than the control (p < .05). The lysozyme, alkaline phosphatase, acid phosphatase (ACP), total antioxidative capacity activities in serum of fish fed 12 g/kg CHMM were higher significantly than those in the control (p < .05), while the total superoxide dismutase (SOD), total antioxidative capacity, catalase, alkaline phosphatase (AKP) and ACP activities in hepatopancreas of fish in 12 g/kg group were all significantly higher than those in the control (p < .05). Regression analysis showed that the relationships between dietary CHMM levels and either FCR, SGR, erepsin, pepsin or lysozyme activities were best expressed by quadratic or cubic regression equations, and the optimal inclusion levels are 11.4, 10.7, 10.7, 8.4 and 10.5 g/kg for maximum FCR, SGR, erepsin, pepsin and lysozyme activities, respectively. Under the present experimental condition, the optimal supplementary level of CHMM in the diet of Japanese seabass is 8–12 g/kg.