The bioaccessibility of soil heavy metals is the solubility of soil heavy metals in synthetic human digestive juice, which is usually determined using in vitro digestion test. To reveal the effects of digestive enzymes on soil heavy metals bioaccessibility, three representative in vitro digestion tests, Simple Bioaccessibility Extraction Test (SBET), Physiologically Based Extraction Test (PBET), and Simple Gastrointestinal Extraction Test (SGET), were chosen. The bioaccessibility of soil Cu, Zn, and Pb in each method were respectively evaluated with and without digestive enzymes, and the differences were compared. The results showed that the effects of digestive enzymes varied with different methods and elements. Because of digestive enzymes addition, the environmental change from acid gastric phase to neutral intestinal phase of PBET did not result in apparently decrease of the bioaccessibility of soil Cu. However, the solubility of soil Zn and Pb were pH-dependent. For SGET, when digestive enzymes were added, its results reflected more variations resulting from soil and element types. The impacts of digestive enzymes on heavy metal dissolution are mostly seen in the intestinal phase. Therefore, digestive enzyme addition is indispensable to the gastrointestinal digestion methods (PBET and SGET), while the pepsin addition is not important for the methods only comprised of gastric digestion (SBET).