BACKGROUND The high frequency and wide distribution of oil spills pose serious threats to the marine environmental ecosystem and human health. Microbial degradation is considered to be the most applicable means to process oil spills. Carbon isotope fractionation was employed as a crucial tool for evaluating the biodegradation of four crude oils by Rhodococcus sp. with Corexit 9500A as oil dispersant. RESULTS Following biodegradation, carbon isotope fractionation of n-alkanes in four dispersed crude oil samples was measured by gas chromatography-isotope ratio mass spectrometry. A relationship between the carbon isotope ratios and residual concentrations of n-alkanes was established based upon the Rayleigh equation, with a correlation coefficient, R-2, ranging from 0.67 to 0.83. The carbon isotope enrichment factors for n-alkanes of the four crude oil samples tended to be 0.04-0.25 parts per thousand in the residuals. The biodegradation level of n-alkanes calculated from the Rayleigh equation agreed with the biodegradation results analyzed by gas chromatography-mass spectrometry. CONCLUSIONS The degree of microbial degradation was positively correlated to the carbon isotope fractionation in n-alkanes. Compound-specific stable isotope analysis is capable of quantifying the biodegradation level of crude oil or oil spills. (c) 2019 Society of Chemical Industry