An eight-week study was conducted to investigate the effects of Kera-Stim®50, a feed additive containing free amino acid mix (KFAAs) on the growth performance, biochemical parameters, innate immune responses, muscle composition, digestive enzyme activity, and muscle fatty acid composition of Nile tilapia (Oreochromis niloticus). Five diets were supplemented with KFAAs at 0% (KFAA0), used as a control group, 0.25% (KFAA0.25), 0.50% (KFAA0.50), 0.75% (KFAA0.75), and 1.0% (KFAA1.0). Compared with the control group, we observed significant improvements in final body weight, weight gain, specific growth rate, average daily gain, protein efficiency ratio, and feed conversion ratio among fish fed diets with increasing KFAA contents in the diets (P < 0.05), especially for the groups that had more than 0.5% KFAAs in the feed. Concurrently, innate immunity parameters such as lysozyme, myeloperoxidase, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase were mostly higher in the groups fed with more than 0.75% KFAAs in the diet (P < 0.05). The activities of digestive enzymes including amylase, protease, and lipase were enhanced by the addition of KFAAs in the feed (P < 0.05). Strikingly, significantly higher contents of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids (especially linoleic acid (C18:2n6c) and α-linolenic acid (C18:3n3) were found in the muscle tissue of fish fed diets containing 0.75% and 1% KFAAs (P < 0.05). We concluded that the addition of at least 0.5% KFAAs can benefit the growth of Nile tilapia, and supplementation with 0.75–1.0% KFAAs can improve the immunity, antioxidant ability, digestibility, and muscle fatty acid composition of Nile tilapia.