Background:: Tetrastigma hemsleyanum (THL) is a traditional medicinal plant with various biological activities, it has been validated that several THL extracts have strong pharmacological activities. Until now, there is no complete and systematic germplasm identification and quality evaluation standard. Objective:: Establishing fingerprint spectra of THL from different sources, evaluating their antioxidant activity and identifying quality markers. Methods:: In this paper, establishing fingerprints and identifying quality markers through UPLC-QTOF- MS/MS. The antioxidant capacity of forty-one batches of THL from multiple regions was determined by 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6- sulphonate) (ABTS), and Ferric reducing antioxidant power (FRAP) assays. Results:: The results indicated that THL methanol extracts showed good antioxidative activity and eight THL samples, including THL aerial parts, showed high antioxidative activity. Besides, Ultra Performance Liquid Chromatography (UPLC) fingerprints of THL from different places were established and chromatographic peaks probably correlated with antioxidant capacity were screened by principal component analysis (PCA) and partial least square regression (PLS) analysis. Conclusion:: The chemical constituents of these peaks were identified as possible quality markers for THL quality evaluation. In this study, twelve effective substances were preliminarily determined, such as isovitexin, L-epicatechin, procyanidin B, sitosterol, etc.