Abstract: The purpose was to optimize the antimicrobial activity in vitro of Chinese chives extract according to various single factor experiments and uniform experiment. Three factors (concentration, temperature, and pH) were taken into consideration. The antimicrobial tests were carried out by the agar diffusion method (oxford cup method) . Inhibition zone diameter was described as the antimicrobial activity indicator. The study found that there was a positive correlation between the concentration of Chinese chives extract and antimicrobial activity. The more concentration, the larger diameter was. Chinese chives extract below 100℃ showed an entire antimicrobial spectrum and outstanding inhibitory effect at 80℃. Under the acid condition, the inhibition effect in vitro of Chinese chives extract wasn’t much different from each other. However, inhibition zone diameter decreased obviously with the pH value increasing under alkalinity condition. Meanwhile, the antimicrobial activity was gradually lower. In the condition of 0.5g/mL, 80℃ , pH4, Chinese chives extract had the strongest antimicrobial activity on Staphyloccocus aureus and the inhibition zone reached 22.4mm.