Purpose: The present study was conducted to assess the ability of probiotic bacteria and yeasts strains to reduce aflatoxin B1 (AFB1) in gastrointestinal simulated conditions. Aflatoxins are potent carcinogenic and immunosuppressive agents. Acute exposure to a high level of aflatoxins leads to aflatoxicosis, which cause rapid death due to liver failure. It is anticipated that consumption of probiotic microorganisms capable of binding aflatoxins can reduce the risk of AFB1 on human health to a certain extent. Methods: For this purpose, the bacteria (1 × 1010 cfu/mL) and yeasts count (2 × 108 cells/mL) and AFB1 concentration (10 ppb) were adjusted. Then, the samples were incubated in the simulated medium, human gastric secretions and small intestine. The concentration of residual AFB1 was determined using enzyme-linked immunosorbent assay (ELISA). The results were statistically analyzed by SPSS 16 software. Results: The native isolated bacteria and yeasts in the simulated gastrointestinal tract condition showed a significant effect on AFB1 reduction (P <0.05). The AFB1 reduction ability of native probiotic microorganisms was strain dependent. The highest binding ability in bacteria belonged to Lactobacillus rhamnosus (31.14%) and at yeasts belonged to Saccharomyces cerevisiae (30.46%). Conclusion: The use of probiotic strains is the appropriate biological method to reduce AFB1 in the human gastrointestinal tract. Probiotic bacteria could help to decrease the harmful effects of AFB1 in humans through enhancing the food safety.