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Aflatoxin B₁ (AFB₁) is the most harmful mycotoxin commonly found in food and feed. Pollution from AFB₁ causes serious economic and health issues worldwide because it causes strong mutagenicity and carcinogenicity in humans and animals. In this study, <i>Pseudomonas aeruginosa</i> was used to degrade AFB₁ in moldy maize, and the safety of this biological method was investigated using genotoxicity and cytotoxicity tests. Using response surface methodology, we established the optimal conditions for degrading AFB₁ by the fermentation supernatant of <i>P. aeruginosa</i>. Under these conditions, the degradation rate of AFB₁ reached 99.67%. Furthermore, the Ames mutagenicity test showed that AFB₁ treated with <i>P. aeruginosa</i> fermentation supernatant for 72 h was not mutagenic. CCK-8 cell assay showed that AFB₁ cytotoxicity was significantly reduced after degradation. Overall, our findings show that the fermentation supernatant of <i>P. aeruginosa</i> may be a good candidate for biodegradation of AFB₁.