The Banana peel is a waste with quite a high nutritional content but rarely utilized, so it needs to be tested for fish farming. This study aimed to evaluate the potential of the Ambon banana (Musa acuminata) peel flour (ABPF) as a prebiotic in the Nile tilapia (Oreochromis niloticus). Fifteen red tilapia fry with an average weight of 2.11 ± 0.18 g were reared for 28 days in a volume of 30 L. This study used a completely randomized design with four treatments and three replications. Experimental fish were fed feed with the addition of ABPF of 0% (control), 1%, 2%, and 3% kg^-1 of feed, twice a day at satiation. The data obtained in this study were: hematological parameters, survival rate, growth performance, feed utilization, the number of lactic acid bacteria (LAB) in the intestine, and digestive enzyme activity statistically analyzed using a one-way ANOVA test and followed with Duncan's test. The results showed that adding ABPF to red tilapia feed significantly affected fish health, especially the levels of hematocytes, total leukocytes, monocytes, and neutrophils were higher than the control treatment to maintain a better survival rate. The final weight and absolute growth of red tilapia were significantly different from the control. The amount of LAB and digestive enzyme activity of red tilapia fed with the addition of ABPF were higher than the control. These results indicate that ABPF may be prebiotic in the Nile tilapia.