Abstract: A Reversed-Phase High Performance Liquid Chromatographic method was developed for determining free Amino Acids in Burley Tobacco. The test was done by using OPA/3-mercaptopropionic Acid as the pre-column derivatising reagent. Chromatographic column was Elitte C18 column (4.6mm×250mm i.d., 5um). Mobile phase A was 18mol/l NaAc (pH7.2) including 0.002%(v/v) triethylamine and 0.3%(v/v) furanidine. Mobile phase B was 100 mol/l NaAc (pH7.2) -Acetonitrile-methanol (v/v=1:2:2). The column temperature was 40℃ and the flow rate was 1.0ml/min. The fluorescence detector was used with 350nm excitation wave length and 450nm emission wave length. The average recoveries of the method ranged 95.3% to 100.7% with the relative standard deviation of 2.32%~9.24%. The method is simple, accurate and has good repeatability. The results of the determination of seventeen kinds of free Amino Acids in burley leaves which were produced by way of different ratio of cake fertilizer and inorganic fertilizer. The results show that Aspartic acid has the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents most of free Amino Acids are increased and then gradually decreased with the increase of organic manure. The contents most of free Amino Acids are very close at 15%: 85% ratio and 30%:70%ratio of cake fertilizer and inorganic fertilizer. The total amount of free Amino Acids is the highest at 30%: 70% ratio of cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at 30%: 70%ratio of cake fertilizer and inorganic fertilizer.