Abstract: Objective:To optimize the ultrasonic-assisted extraction conditions of polyphenols from the stems of Trapa bispinosa Roxb.and evaluate the antioxidant and antimicrobial activities of extracted polyphones in vitro. Methods:The ultrasonic-assisted extraction process was optimized using orthogonal test. The antioxidant activity was evaluated by determination of DPPH, ABTS radical scavenging and ferric reducing antioxidant potential assay (FRAP). The antimicrobial activity against six spoilage microorganisms was tested by the method of disk diffusion. Results:The optimal extraction processing parameters were ethanol concentration of 60%, pH of 1, extraction temperature of 70℃, extraction time of 35 min, solid-to-liquid ratio (g/mL) of 1:35.Under these conditions, the highest extraction yield value reached 67.31 mg GAE/g DW. Furthermore, the polyphenols obtained from Trapa bispinosa Roxb presented significant antioxidant activity through DPPH, ABTS and FRAP methods with the IC₅₀ values of 228.34, 220.57 and 152.00 μg/mL, respectively. The minimal inhibitory concertration (MIC) against Staphyloccocus aureus, Bacillus cereus, Escherichia coli, Shewanella putrefaciens, Penicillium glaucum and Saccharomyces cerevisiae were 6.25, 3.13, 0.20, 0.39, 1.56 and 0.78 mg/mL, respectively. Conclusion:The polyphenols from the stems of Trapa bispinosa Roxb. showed excellent antioxidant and antimicrobial activities, which could be used as a source of potential antioxidant and antimicrobial agents.