Dihydromyricetin (DMY) was separated and purified from Ampelopsis using water extraction and recrystallization. The crystal morphology was analyzed by scanning electron microscopy and the cellular antioxidant activity (CAA) was determined by CAA assay. Meanwhile, in vitro bile salt-binding ability of DMY was determined by in vitro simulation of the human gastrointestinal environment. The results showed that separation and purification techniques with multiple recrystallizations produced high-purity DMY, and the yield was 43%. An EC50 of 6.17 ± 0.08 μmol/L and CAA of 74.90 ± 0.90 μmol QE/100 μmol were obtained, which indicated that DMY exhibited strong CAA. DMY also exhibited relatively strong binding to sodium cholate, sodium glycocholate, and sodium taurocholate, with binding capacity of 1.24 ± 0.02, 0.97 ± 0.06, and 1.06 ± 0.002 μmol/m1, respectively, with strongest binding capacity for free sodium cholate.