This study evaluated the effects of dietary supplementation with different doses (0, 10, 20, 40, and 80 g kg−1) of powdered avocado seed (AS) on the growth performance, immunological response, and gene expression of Nile tilapia (Oreochromis niloticus) reared in a biofloc system over 8 weeks. A total of 300 Nile tilapia fingerlings (average weight 14.67 ± 0.07 g) were randomly assigned to five treatment groups, each with three replicates, and 20 fish per tank. The results demonstrated significant improvements (p < 0.05) in growth and immune response in AS-supplemented fish, particularly in those fed the 10 g kg−1 AS diet (AS10), which showed the most notable increases. In contrast, fish fed higher AS doses (AS20, AS40, and AS80) exhibited no statistically significant differences compared to the control group (p > 0.05). Additionally, the AS10 group exhibited a significant upregulation (p < 0.05) in the mRNA expression of key immune-related genes (IL-1, IL-8, and LBP) and antioxidant-related genes (GST-α, GPX, and GSR) in both liver and intestinal tissues, indicating enhanced immune and antioxidant responses. The highest expression levels were found in the AS10 group. These findings suggest that the inclusion of 10 g kg−1 powdered avocado seed in the diet substantially enhances growth, immune function, and gene expression in Nile tilapia reared in a biofloc system. The results highlight avocado seed as a promising feed additive for improving the sustainability of Nile tilapia aquaculture.