We attempted to determine the bioethanol content of E3 gasoline by applying ASTM D6866 method B. In the pre-treatment process using accelerator mass spectroscopy(AMS), the graphite samples were prepared from E3 gasoline. Three portions of the same graphite sample were measured, and the contents agreed within the measurement error of AMS. The graphite samples prepared from eight portions of the same E3 gasoline sample were measured, but the accuracy was insufficient. There are many kinds of hydrocarbon compounds in the gasoline and their boiling points are different. The content of bioethanol was found to decrease with vaporization when E3 gasoline was placed in open air. A very small amount of E3 gasoline is pre-treated for AMS and the volatile loss cannot be ignored. It seems that the content change of bioethanol was caused by vaporization of E3 gasoline during the pre-treatment process.