AbstractTetrastigma hemsleyanum is traditionally used as a folk medicine and functional food in China. Its extracts have been confirmed to have many bioactivities. However, the effect of extracting temperature on its bioactivity has not been reported. In this research, the total flavonoids content (TFC), total polyphenol content (TPC), antiproliferative, antioxidant, and anti‐inflammatory activities of ethanol extracts and water extracts (extracted at 55, 70, 85, and 100°C) were observed. The results indicated that ethanol extracts showed better antioxidant activity with DPPH EC50 of 504.1 ± 3.8 μg/ml and ABTS EC50 of 851.4 ± 3.9 μg/ml. A better antiproliferative activity on HepG2, PC12, Caco‐2, and Hela cells was observed on ethanol extracts. The results of anti‐inflammatory activities also indicated that all of the extracts can reduce the NO production of LPS‐stimulated macrophage with dose‐independent manner. In summary, the results showed that the antiproliferative, antioxidant, and anti‐inflammatory activities of water extracts decreased with the increasing temperature to some extent.