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The purpose of this study was to evaluate the ability of Lactobacillus rhamnosus to bind patulin (PAT) in the buffer solution and apple juice. The binding of L. rhamnosus to PAT was reversible, which improved the stability of the bacterial complex. The ability to bind PAT can be enhanced with the inactivation of the strain by high temperature and acid treatment. Acid-treated bacteria had the highest PAT binding rate of 72.73±1.05%. The binding rates of acid and high temperature (121 °C) treatments were increased by 21.37% and 19.15%, respectively. L. rhamnosus showed the best detoxification ability to PAT at 37 °C, where the binding rate reached 50.9±1.03%. When the dose of inactivated bacteria powder was 0.02 g ml−1, the minimum concentration of PAT in apple juice was 0.37 µg ml−1. The addition of the L. rhamnosus inactivated powder did not affect the quality of the juice product and effectively bound the PAT in apple juice.