Abstract: Objective:To optimize the extraction procedure of liposoluble constituents in Bletilla striata flower by response surface methodology. And study on its bioactivity such as antibacterial activity, antitumor activity and antioxidant activity. Methods:Response surface methodology was used to design, optimize and verify the extraction procedure of liposoluble constituents in B. striata flower on the basis of single-factor experiments, so as to improve the yield of liposoluble component. It was evaluated for antibacterial activity by paper diffusion method, and antitumor activity by CCK8 method, while its antioxidant activity in vitro was evaluated by removal ability of DPPH·, ABTS⁺·and hydroxyl free radicals.Results:The results of optimum extraction condition were as follows:Extraction time 2.94 h, 2 times of extraction, the material-liquid ratio 1:40 g/mL. Under these conditions, the predicted values of the yield of liposoluble components could reach 1.416%, the average yield of the verification test was 1.401%±0.112%, which was 28.49% higher than before. Bioactivity tests showed that:Liposoluble components in B. striata flower had significantly inhibitory effect on Bacillus subtilis, Staphyloccocus aureus, Escherichia coli and Candida albicans four kind of pathogenic bacterias, as well as the inhibitory rate of A₅₄₉ cells had raised 32.115% determinated by CCK-8 method. It also had inoxidizability and its EC₅₀ was 1.63, 0.47 and 1.25 mg/mL for DPPH free radical, ABTS free radical and hydroxyl radical respectively. Conclusion:The optimized extraction procedure was reasonable and feasible. The liposoluble components in B. striata flower had significant biological activity and potential medicinal value.