Patulin (PAT) is a toxic metabolite produced by several filamentous fungi common to fruit- and vegetable-based products, which results in a potential food safety hazard. Over the past few years, there have been reports stating that PAT was removed by yeast from some fermented products. However, the interactions between PAT and yeast during fermentation are still unclear. In this paper, we focused on the interactions between PAT and S. cerevisiae CITCC 93161 in aqueous solutions during fermentation. The results demonstrated that PAT affected the yeast cells in a time- and concentration-dependent manner, but the yeast exhibited a very strong tolerance to the toxicity of PAT. The yeast cells could completely degraded PAT through the exo- and endo-enzymes synthesized during yeast fermentation, of which the latter exhibited higher activity. Moreover, the PAT biodegradation ability of yeast was not induced by the addition of such toxics to the culture media. Additionally, E-ascladiol was tentatively identified as the product of PAT biodegradation.