This study aimed to optimize trienzyme-assisted extraction (EAE) conditions for total polyphenols (TP) from Ulmus pumila barks (UPB). Response surface methodology (RSM) was used to optimize EAE conditions including pH, temperature, and time. The extraction efficiency of three extraction procedures on the TP yield, antioxidant activities and chemical composition of UPB extracts was also compared and characterized. Our results showed that the maximum extraction yield of TP was 16.04±0.38mg gallic acid equivalents/g dry weight (GAE/g DW) under the optimum EAE conditions (pH=4.63, 52.6°C and 62min). Meanwhile, the EAE gave a higher extraction yield of TP and then a greater in vitro antioxidant capacity compared with those obtained from both ultrasound-assisted extraction (UAE) and conventional heat extraction (CHE). In addition, seven polyphenolic compounds were validated by high-performance liquid chromatography analysis in the extracts at the optimized conditions. The results of this study further confirmed that EAE could be explored as a state-of-the-art environmentally friendly technology for recovering optimum amounts of antioxidant polyphenols from plant sources.