A Reversed-Phase High Performance Liquid Chromatographic method was developed for determining free Amino Acids in Burley Tobacco. The test was done by 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 18mmol/L NaAc (PH=7.2) including 0.002% (v/v) triethylamine and 0.3% (v/v) furanidine. Mobile phase B was 100 mmol/L NaAc (PH=7.2) -Acetonitrile- methanol (v/v=1:2:2). The column temperature was 40°C 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 most contents of free amino acids are increased and then gradually decreased with the increase of organic manure. The most contents of free amino acids are very close at the ratio of 15%:85% ratio and 30%:70% between cake fertilizer and inorganic fertilizer. The total content of free amino acids is the highest at the ratio of 30%: 70% between cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at the ratio of 30%:70% between cake fertilizer and inorganic fertilizer.