Herbal supplements are suitable for improving fish health and combating diseases in fish culture. However, the mechanism of action of many herbal mixtures remains unclear. This study was conducted to evaluate the effects of traditional Chinese medicine (TCM; a mix of Astragalus membranaceus, Angelica sinensis, and Crataegus hupehensis at a ratio of 1:1:1 on a weight basis) on growth, immune response, and disease resistance in Nile tilapia Oreochromis niloticus. Experimental fish (mean ± SE weight = 57 ± 1 g) were divided into two groups: a control group and a TCM (10 g/kg) group. After 4 weeks of the experimental trial, a significant increase in weight gain and specific growth rate and a lower feed conversion ratio were observed in fish fed a TCM-supplemented diet compared with control fish. Similarly, the immune response of Nile Tilapia in the TCM group showed enhanced lysozyme, superoxide dismutase, catalase, and immunoglobulin levels compared with the control fish. In comparison with the control, fish fed TCM showed significant up-regulation of β-defensin, lysozyme, heat shock protein 70, superoxide dismutase, and catalase genes in the intestine and head-kidney tissues. After a Streptococcus agalactiae challenge, survival of Nile Tilapia in the TCM group was 70% compared with 35% in the control. These results indicate that the TCM mixture in this study can elevate the immune response and disease resistance of Nile Tilapia.