Probiotics are dietary microbial supplements used as alternatives to antibiotics in aquaculture. The present study was performed to evaluate the use of Bacillus amyloliquefaciens as a probiotic bacteria in the diets for Nile tilapia, Oreochromis niloticus. A total of 180 fingerlings (9.4 ± 0.3 g) were divided into three groups (G1–G3 of 20 fish per group) in triplicate. G1 was fed a basal diet with no probiotic as the control group, while G2 and G3 were fed B. amyloliquefaciens spores at concentrations of 1 × 104 and 1 × 106 colony-forming units per gram (CFU/g) of feed, respectively. After 30 days, G3 showed significant improvements in body weight (BW), weight gain (WG), specific growth rate (SGR) and food conversion ratio (FCR) compared with G1 and G2. At the end of the feeding period (60 days), both G2 and G3 had significantly higher FBW, WG, SGR, FCR and protein efficacy ratio compared with the control. The increase in the probiotic level in the diet increased the heights of the intestinal villi, especially in the proximal part of the intestine. In G3, the proximal and distal parts had significantly higher numbers of mucus-secreting cells (goblet cells) than in G2 and G1. There were significantly more intra-epithelial lymphocytes (IEL) in the intestine in G3 compared with the control in all parts of the intestines, whereas G2 had significantly more IELs than the control in only the distal part of the intestine. B. amyloliquefaciens had no harmful effects on glucose levels, and increased the erythrocyte and leukocyte counts, hemoglobin content and hematocrit. High levels of the probiotic (G3) had better effects than lower levels (G2) on serum total protein and globulin levels. The protein contents of the whole body in G3 and G2 were higher than those of the control group. The highest body fat content was recorded in G3, followed by G2. These results indicate that the use B. amyloliquefaciens as a feed supplement is beneficial to O. niloticus.