The aim of this study was to quickly discover multifunctional bioactive flavor peptides from the enzymolysis extracts of Lentinula edodes through bioinformatics, molecular docking and molecular dynamics simulation, and to evaluate the synthesized and in vitro activity of the screened peptides. The biological activity, toxicity, allergenicity, solubility and stability of 272 L. edodes enzymolysis flavor peptides were predicted, and 15 potential bioactive peptides were screened. By molecular docking prediction of 15 potential bioactive peptides, it was found that angiotensin-converting enzyme (ACE)’s Glu403, Glu411, Arg124, His387 and dipeptidyl peptidase IV (DPP-IV)’s Glu206, Arg125, Lys554, Asp545, Arg560 might play a key role in binding to potential bioactive peptides. Among them, DAPLPHPNR and GSEDPLPGAK showed stronger bonding energy, and the stability of their complexes was further verified by molecular dynamics simulation. In addition, the two peptides were synthesized by chemical method, and in vitro experiments showed strong ACE and DPP-IV inhibitory activities, while also having different antioxidant effects. This work provides an efficient method for screening and developing multifunctional flavor peptides in edible fungi.