Objective To establish an effective and concise method for determining the bioavailability of lead and apply it to the evaluation of lead bioavailability in rice.Methods The study investigates the effects of pH, digestive enzymes, and digestion time in in vitro digestion on the release of lead in the oral phase, gastric phase, and small intestinal phase. Meanwhile, it determines the bioaccessibility and bioavailability of lead with Caco-2 cells.Results The results show that the optimal in vitro digestion conditions are pH 1.5 with pepsin addition and digestion for 2 hours in the gastric phase and pH 7.0 and digestion for 4 hours in the small intestinal phase. The ratio of alkaline phosphatase activity, transmembrane resistance, and the surface microvillus structure of Caco-2 cells are used to establish the in vitro simulation model of small intestinal phase digestion. Simultaneously, the bioaccessibility of lead in raw and cooked rice is 64.94%~78.70% and 57.06%~72.20%, respectively in the gastric phase and 22.94%~33.77% and 21.23%~27.24%, respectively in the intestinal phase. Meanwhile, the bioavailability in the small intestinal phase is 2.37%~6.28% and 1.57%~4.09%, respectively.Conclusion The method established in this study can be used to evaluate the bioavailability of lead in rice. In addition, cooking can reduce the bioaccessibility and bioavailability of lead in rice during gastrointestinal digestion and absorption, and only a small fraction of lead in rice can enter the body through the circulation.