Abstract: Objective: To study the spectrum-effect relationship between the extracts of Tetrastigma hemsleyanum and its antioxidant activity, and to clarify the quality markers of its antioxidant activity. Methods: The extract was prepared and separated by macroporous resin elution, six different elution sites were obtained using ethanol at concentrations of 10%, 30%, 50%, 70%, 90% and 100%. The fingerprints of the Tetrastigma hemsleyanum extract and each elution site were established by UPLC-Q-TOF-MS and the characteristic peaks were screened. The free radical scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was used as an antioxidant index to investigate the antioxidant activity of the extract and each elution site of Tetrastigma hemsleyanum. The Pearson correlation coefficient method and orthogonal partial least squares (OPLS) method were combined to analyze and study the spectrum-effect relationship between the characteristic peaks and the antioxidant activity, to screen the quality markers, and to analyze and identify the chemical compositions. Results: Under the positive and negative ion modes, 57 and 92 characteristic peaks were identified in the extract and each elution site of Tetrastigma hemsleyanum, respectively. The combined results of Pearson correlation coefficient and OPLS analysis showed that 14 ion peaks contributed more to the antioxidant activity, which was vanillin (P2), catechin (P13, N19), rutin (P27, N54), and isoquercitrin (P28), kaempferol-3-O-rutinoside (P33, N61), proanthocyanidin B (N18), proanthocyanidin C (N22), and ion peaks N23, N25, N26, and N29, respectively. Conclusion: UPLC-Q-TOF-MS fingerprints of Tetrastigma hemsleyanum extract and each elution site were established, and the quality markers of Tetrastigma hemsleyanum antioxidant activity were revealed to provide technical support for the formulation of quality standards and resource development and utilization of Tetrastigma hemsleyanum.