Stem‐leaf saponins (SLSs), the total saponins from aerial part of P. notoginseng, are by‐products of notoginseng, a famous traditional Chinese medicine. SLSs have been used as a health functional food in China, but its mild effects limited clinical applications in diseases. Inspired by steaming of notoginseng to enhance its pharmacological activity, a steaming protocol was developed to treat SLSs. SLSs were steamed at 100, 120, and 140°C for 1, 2, 3, and 4 h, respectively. The ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight MS and ultra‐performance liquid chromatography–tandem triple quadrupole mass spectrometry were applied to analyze the dynamic changes in chemical compositions. The anti‐acetylcholinesterase activity of steamed SLS were assessed in vitro by directly determining the metabolic product of acetylcholine/choline. The components of SLSs were significantly changed by steaming. A total of 117 saponins and aglycones were characterized, and 35 of them were newly generated. The anti‐acetylcholinesterase activity of steamed SLSs gradually increased with the extension of steamed time and the increase of steamed temperature and reached the maximum after 140°C for 3 h. Furthermore, ginsenosides Rk1 and Rg5, the main components of steamed SLSs, showed dose‐dependent anti‐acetylcholinesterase activities with half maximal inhibitory concentration (IC50) values of 26.88 ± 0.53 μm and 22.41 ± 1.31 μm that were only 1.8‐ and 1.5‐fold higher than that of donepezil with IC50 values of 14.93 ± 4.17 μM, respectively.