The aim of this study was to optimize the purification process for total flavonoids from the aerial parts of Tetrastigma hemsleyanum Diels & Gilg (TFATH) by using macroporous resin and to evaluate the protective effects of TFATH against carbon tetrachloride-induced acute liver injury in mice. The effects of different loading conditions and elution conditions on the purification results were investigated using single-factor experiments, and the antioxidant activity of the aerial parts of Tetrastigma hemsleyanum Diels & Gilg was determined before and after purification. TFATH were qualitatively analyzed using UPLC-ESIQTOF-MS2, and an acute liver injury model induced by carbon tetrachloride was established. The results showed that the purity of flavonoids in the purified samples increased from 8.99% to 31.38%, and the antioxidant capacity also significantly improved. Twelve types of flavonoids were identified, primarily including (iso) vitexin, (iso) orientin, and their glycosides. The animal experiment results showed that, compared with the model group, the liver index and serum ALT and AST activities of TFATH-treated mice significantly decreased by 9.74%, 12.91%, and 33.98%, respectively, and the degeneration, necrosis, and inflammatory infiltration of hepatocytes significantly improved. In liver tissue, GSH content and CAT activity increased by 54.14% and 50.88%, respectively, whereas the MDA content significantly decreased by 66.53%. In addition, with the intervention of TFATH, the expression levels of TNF-α, IL-1β, IL-6, caspase-3, Bax, and Keap1 were significantly downregulated, whereas the expression levels of Bcl-2, Nrf2, HO-1, NQO1, and GCLC were upregulated. In summary, TFATH comprehensively ameliorates acute liver injury induced by carbon tetrachloride by activating the Keap 1/Nrf 2 signaling pathway, modulating oxidative stress, alleviating inflammatory responses, reducing the expression of proinflammatory cytokines, and inhibiting hepatocyte apoptosis. The results provide a theoretical reference for the development and utilization of resources from the aerial parts of Tetrastigma hemsleyanum Diels & Gilg.