The aim of this study was to check the effect of multiple temperatures and various protein diet formulas on liver enzymes, gonadotropins, and growth hormone (GH) using African catfish Clarias gariepinus. Clarias gariepinus was exposed to multiple temperatures (T24°C, T28°C, and T32°C) and various protein diet formulas: D1 (fishmeal-based diet), D2 (soymeal based diet), and D3 (pea-meal based diet). Tilapia commercial feed (D4) was used as reference diet. A total of 720 individuals with an average weight (101–104 g) were stocked at a density of 20 individual fish per tank in 12 tanks of three replicates. Liver enzymes, gonadotropins, GH and fish fecundity were measured after 16 weeks. The results revealed that liver enzyme like glutamate-oxaloacetate transaminase was significantly (p 0.05) at any experimental condition. Follicle-stimulating hormone and luteinizing hormone were significantly (p < 0.05) increased at T28°C: D1 diet and T32°C: D1 diet, respectively. GH was significantly (p < 0.05) increased by T28°C: D1. The relative weight of the ovary of C. gariepinus was significantly (p < 0.05) increase at T32°C: D1, while the testis relative weight was increased with T24°C: D3. The result from this study revealed that there is a direct relationship of temperature on fish fecundity, enzymes, and reproductive hormones in C. gariepinus. The temperature of 28°C along with fishmeal or soy-meal positively improved the fecundity and health of fish.