The polybasic acids and higher fatty acids in tobacco were methyl esterified under two derivative conditions: heating-refluxing and placing at room temperature. The reaction mixtures were analyzed with gas chromatography (GC), the results were compared by means of t test. The experimental results revealed that: 1) The results existed significant difference between the two derivative conditions, and refluxing derivation was preferred in the case of precise quantification. 2) In the established method, polybasic acids and higher fatty acids in tobacco were derived into methyl esters by refluxing with sulfuric acid/methanol solution, then analyzed with GC. The recoveries of the method ranged from 90.72 % to 98.92 % with RSDs below 3 %. The derivative method of refluxing features high accuracy and good repeatability, is suitable for determining polybasic acids and higher fatty acids in batches of tobacco samples.