The biological activity and optimum conditions for threeleaf cayratia (Cayratia trifolia L.) wine fermentation using Saccharomyces cerevisiae HG1.3 were studied. The tests on the effect of fermentation conditions, including initial total sugar content (20, 22, 24 °Brix), pH (4.0, 4.5, 5.0), inoculum concentration (103, 105, 107 cells/mL), and temperature (30, 35, 37, 39, 41 °C) indicated that the ethanol contents of threeleaf cayratia wine were 5.19–12.82% (v/v). The results showed that appropriate fermentation conditions were 35 °C, 20 °Brix, 105 cells/mL yeast cells, and 6 days. The ethanol content of the final product was 11.68% (v/v) and the methanol, SO₂, hydrocyanic acid, and aldehyde concentrations were acceptable according to Vietnam National Standard 7045:2002. The total polyphenol content and antioxidant activity of threeleaf cayratia juice (0.66 mg gallic acid equivalents (GAE)/mL and 54.7%, respectively) and wine (0.60 mg GAE/mL and 57.4%, respectively) were not significantly different.