Abstract: The content of fatty acid methyl ester in mixed fuel of diesel and biodiesel is an important specification for the product. Using IR spectrum method to determine the total amount of fatty acid methyl ester, the weakness is that it cannot well distinguish the absorbency of ester functional group comes from fatty acid methyl ester or unconverted oil feed, mainly triglycerides. Thus, investigation of using high temperature gas chromatographic method to separate mixed biodiesel fuels adding various kinds of oil feed was carried out, and a method for the determination of triglycerides in mixed biodiesel fuel was established with tricaprin as internal standard. The determination limit (mass) was 0.01%. When the content of triglycerides in fuel was over 0.1%, the recovery rate reached more than 91% and relative standard deviation was less than 6%. Above results indicates that this method could meet the demand of conventional analysis, it could be used as a supplemental method to help distinguish the source of ester functional group for IR spectrum method in need.