Abstract: The extraction and analysis method of total polyphenol from black pepper (Piper nigrum L.)was established to explore the distribution in black pepper fruit and antioxidant activity of total polyphenol, which would be contributed to the further study on total polyphenol in black pepper. Black pepper fruit as the main raw material, through single factor and orthogonal experiment, the influence of ethanol concentration, extraction time, solid-liquid ratio, extraction temperature and so on four factors on pepper total polyphenol extraction yield was analyzed. The total polyphenol content in extract of black pepper was analyzed using Folin-Ciocalteu colorimetry with gallic acid as standard. The total polyphenol content in pepper peel, seed and stem was also measured. The antioxidant potential of the total polyphenol extract from black pepper was evaluated for their ability to scavenge 1,1’-diphenyl-2-picrylhydrazyl( DPPH) free radical, and half maximal inhibitory concentration (IC50) was calculated as antioxidant activity evaluation index. The results showed that the optimal condition for total polyphenol extraction was ethanol of 55%，solid-liquid ratio of 1:30 (g/mL), temperature of 90℃ and time of 4 h, respectively. The total polyphenol content in black pepper was 6.556 mg/g. The highest content of total polyphenol (12.208 mg/g) was detected in black pepper stem, followed by peel (12.105 mg/g), while the lowest was in seed (2.891 mg/g). The total polyphenol content in black pepper was a significant negative correlated with IC50 value (P<0.05). Compared with vitamin C, the total polyphenol extract from black pepper had more powerful free radical scavenging capacity. Total polyphenol might be the main component contributing to antioxidant activity of pepper. Peel and stem of black pepper could be used as good source of natural antioxidant.