The present study was conducted to replace fish meal with alternative plant protein sources in aquafeeds. Therefore, soybean meal (SBM), corn gluten meal (CGM), and mix of SBM + CGM were used as alternatives to fish meal (FM) with or without betaine (B) in Nile tilapia diets. Positive control diet using FM and six other diets were formulated to contain SBM, SBM + B, CGM, CGM + B, SBM + CGM, or SBM + CGM + B. Dietary B improved fish growth and body protein content in comparison to the control and equal to fish fed FM-based diet. Reduced FCR and body fat content were observed in the case of fish fed FM, SBM + B, CGM + B, or SBM + CGM + B diets. The survival rate was significantly increased in the group of fish fed SBM + CGM + B when compared to fish fed CGM diet (P = 0.013) while no significant differences were reported among the other groups (P > 0.05). Markedly increased intestinal villi length and the number of goblet cells were found in FM, SBM+B, CGM+B, and SBM+CGM+B groups (P < 0.05). Hemoglobin, blood total protein, and IGF-1 gene expression were significantly (P < 0.05) increased in fish fed FM, CGM+B, SBM+CGM, and SBM+CGM+B diets, while fatty acid synthetase (FAS) and lipoprotein lipase (LPL) were downregulated. The highest RBCs, WBCs, and globulin were observed in fish fed SBM + CGM + B diet, while the lowest number was found in the fish fed CGM diet. To sum up, B supplementation in tilapia diets acted synergistically with SBM, CGM, or both SBM + CGM to improve the growth, intestinal morphometrical indices, hemoglobin, and total protein and IGF-1 as well as decreased FCR, FAS, and LPL.