Abstract: Polyphenol content from neem leaf was measured at 750nm wavelength with gallic acid as standard by the using Folin-Ciocalteu method. The extraction conditions of total polyphenol from the neem leaf were optimized by solvent refluxing method. The interactions between the variables, and three response variables such as extract temperature, time and ethanol concentration affected on the extraction of polyphenol from neem leaf were investigated by the single factor experiment and response surface method. And the extraction process was optimized. Moreover, antioxidant activity of polyphenol in leaves of neem was evaluated by in vitro. Experimental results show: (1) the optimum extraction conditions of polyphenol from the neem leaf were temperature 77 ℃, extraction time 80 min and ethanol concentration 50 %; (2) the influence order of response variables was extraction temperature, extraction time, ethanol concentration; (3) the average extract ratio of polyphenol in the neem leaf was 3.12 %; (4) under the same concentration, the antioxidant activity of polyphenol extracted from neem leaf was stronger than that of Vc, and the IC50 values of phenolics scavenging activity on DPPH was approximately 4.6 mg/L. It was less than that of Vc which was approximately 9.8 mg/L. (5) the 23 mg/L of IC50 values of neem leaf phenolics scavenging activity on OH was less than that of Vc which was about 85 mg/L.