Abstract: To study steam distillation extraction technology of volatile oil from Hypericum perforatum L. flowers,the type of parameters soaking time,solvent to solid sample ratio and extraction time were investigated in single factor experiments with extraction yield of volatile oil as evaluation index.The best extraction processes were obtained with three factors and three levels of response surface methodology as follows: soaking time 19 h,solvent to solid sample ratio 9∶ 1 m L / g and extraction time 10 h respectively,leading to an extraction yield of volatile oil 1.03 m L /100 g,and the experimental value are basically close to the theoretical expectation value 1.10 m L /100 g,with only 2.73% deviation. Combining with the antibacterial activity tests,the antimicrobial cycle diameter of volatile oil from Hypericum perforatum L. flowers were 9,10,12 mm for Escherichia coli,Staphyloccocus aureus and Bacillus subtilis,which suggested volatile oil from Hypericum perforatum L. flowers has a certain antibacterial effect.