A method for determining 20 free amino acids in tobacco leaf was established by using 2,4-dinitrofluorobenzene (DNFB) as a derivative reagent, 0.015 mol/L disodium hydrogen phosphate and potassium dihydrogen phosphate as buffer solution and 50% aqueous acetonitrile as mobile phase, and reversed phase HPLC. Seven tobacco leaf samples were detected. The results showed that: 1) The average recoveries of the 20 amino acids were from 94.2% to 118.4% with the RSDs of less than 3%. 2) Methionine and isoleucine were not detected in four flue-cured tobacco samples, the content of proline was the highest, and the contents of total free amino acids in two upper leaf samples were higher than those in two cutter samples. 3) The content of total free amino acids in the sample of Brazil burley tobacco, BCRA, was the highest, and its aspartic acid content was significantly higher than that in other samples. 4) The phenylalanine content in the sample of Maryland tobacco was the highest, and asparagine content was obviously higher than that in other samples, and proline content was slightly higher than that in the sample of Greece oriental and significantly higher than that in the other five samples.