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The purpose of this research was to understand the influence of carbon source variation in the biosurfactant production by Azotobacter vinelandii as well as its potential application on remediation of hydrocarbon-contaminated soil by soil washing. The growth microorganism, biosurfactant quantity in exopolysaccharide and fatty acid fraction were examined. The quality of biosurfactant was analysed by emulsification index. The results show that biggest production was occured at the 48th hour with μmax1 = 0.416/hour, μmax2 = 0.093/hour and Ks1 = 6.55 g/L, Ks2 = 4.69 g/L for glucose and molasses substrat respectively. Biosurfactant product of Azotobacter vinelandii in glucose 1.5%, 2%, 2.5%, and 3% showed emulsification index as much as 100%. While, emulsification index of the biosurfactant produced with molasses reached 100% within 1%, 1.5%, and 2% molasse concentration. This results showed that molasses has potential utilization as carbon source in biosurfactant production. In soil washing application, Biosurfactant and Tween 80 were used as emulsifier to removed Total Petroleum Hydrocarbons (TPHs) by contacting in erlenmeyer flask at rotary shaker. The results showed that highest removal achieved by using biosurfactant. The Highest TPHs removal reach 48.89% which occured at 100 rpm for 40 minutes.