Abstract: This paper used the extracts from the seed meal of Camellia oleifera by n-butanol as raw material, applied separation and purification technology of macroporous resin column chromatography, normal phase silica gel column chromatography and high performance liquid chromatography to isolate the substances with antibacterial activity. The results showed that, used 60% ethanol on macroporous resin column chromatography, then gradient eluted with ethyl acetate, methanol, formic acid volume ratio of 9：1：0.1, 8：2：0.1, 7：3：0.1 on normal phase silica gel, purificated with 30% methanol as mobile phase of high performance liquid chromatography, the antibacterial substance with very high purity could be isolated from the seed meal of C. oleifera by n-butanol extracts.