Gamma-aminobutyric acid (GABA), a four-carbon non-proteinogenic amino acid, exhibits extensive antioxidant, anti-inflammatory, and anti-fatigue properties in both in vivo and in vitro studies. This study optimized the fermentation conditions for GABA-enriched camel whey protein (CWP) and investigated its anti-fatigue effects and gut microbiota modulation mechanisms using a BALB/c mouse cancer-fatigue model. The results demonstrated that CWP supplemented with glucose and 2 g/L monosodium glutamate (MSG), when fermented with 1.5% Streptococcus thermophilus 1303 and 2.5% Lactobacillus brevis ATCC 367 at 40℃ for 48 h, yielded the highest GABA content (1.29 g/L). Additionally, it exhibited significant anti-fatigue effects in mice, including prolonged forced swimming time, enhanced open-field test performance, reduced levels of blood lactate, urea nitrogen and inflammatory cytokines, and increased glycogen and muscle ATP content. Furthermore, fermented CWP positively modulated amino acid absorption, abundance of Bacteroidetes and Firmicutes and short-chain fatty acid (SCFA) metabolism. Correlation analysis revealed significant associations between altered gut microbiota composition, fatigue-related parameters, blood amino acid profiles and SCFA levels. This research supports the development of fermented dairy products with high anti-fatigue potential and provides a novel nutritional intervention for cancer patients.