Ampelopsis grossedentata leaf is rich in polyphenols, and now is regarded as a new food material with high value for utilization. Two typical kinds of calluses were induced from Ampelopsis grossedentata young leaves. The excellent calluses were successfully obtained by hormone optimization and successive screening, which were further used to develop the cell suspension culture system of Ampelopsis grossedentata leaf. The main polyphenols in the suspensively cultured cells were identified, and their antioxidant activities were evaluated in the meantime. Seven polyphenols were identified in the extracts of calluses by the UPLC-Q-TOF MS/MS and HLPC detection, while ten polyphenols were identified in the suspensively cultured cells. Three polyphenols including proanthocyanidin B1, (+)-catechin, epigetechin gallate were found both in calluses and cells with high concentration, among which epicatechin gallate was the most abundant compound, whose contents reached 5020.965μg/g and 1044.725μg/g in calluses and cells, respectively. In addition, total polyphenols extracts from the suspensively cultured cells displayed strong antioxidant activities, with IC 50values of 30 681 μg/mL and 3.685μg/mL respectively in the ABTS⁺ and DPPH free radical scavenging tests. What's more, both the DPPH radical scavenging and total reduction ability of the polyphenol extracts were significantly stronger than vitamin C under the same mass concentration, which were mainly attributed to epicatechin gallate and (+)-catechin. It was hoped that this study would provide a reference for the culture of Ampelopsis grossedentata leaf cells and the regulation of its polyphenolic secondary metabolite synthesis.