In this study, the influence of in vitro gastrointestinal digestion on the Perilla frutescens leaf extract (PFLE) were measured. Results revealed that total phenolic content (TPC) and total flavonoid content (TFC) were significantly decreased after simulated digestion (ca. 53% of phenolics and 40% of flavonoids). The IC 50 value of DPPH• scavenging activity and ABTS + scavenging ability increased by 23% and 56%, respectively, while ferric reducing antioxidant power reduced by 53%. For the inhibition ability on α-glucosidase, acetylcholinesterase, and MCF-7 cell proliferation, their IC 50 values increased by 360%, 197%, and 25%, respectively. Three phenolic acids and one flavonoid in PFLE were quantified by high-performance liquid chromatography. Overall, although significant losses of the active components and biological activities occurred during in vitro gastrointestinal digestion, it still showed the potential as an oral agent for treatment and prevention of oxidative stress, cancer, diabetes, and Alzheimer's disease. Practical applications As an important annual herbaceous plant with rich biochemical compounds and many biological functions, Perilla frutescens leave is widely used in the food and traditional Chinese medicine. However, the dynamic changes of its active compounds and activities during the digestion process are unclear. In this study, the digestion results in significant loss of the active ingredients and biological activities of P. frutescens leaf extract (PFLE), particularly in the gastric digestion. In addition, PFLE remains to show certain antioxidant activity, α-glucosidase inhibitory ability, acetylcholinesterase inhibitory ability, and MCF-7 cell proliferation inhibitory ability after digestion.