Biodemulsifier is one of the green demulsifier which could be hopeful used in crude oil industry. In this paper, pretreatment with dichloromethane and water extraction was used to extract active demulsification substance from the demulsifying bacteria Alcaligenes sp. S-XJ-1. The crude extract were consist of four amino compounds and one composition of saccharide by thin layer chromatography. It was established experiment condition of silica gel column chromatograph for crude extract purification. Demulsification ratio of the purified sample G4 was 70.8% after 48h. The purified active substance was a peptide compound by further analysed with infrared spectrum and amino acid analysis.