A simple and reliable spectrofluorimetric method has been developed for the determination of tramadol HCl in the pharmaceutical preparation and biological fluids. The fluorescence of the product was found to have excitation at max 272 nm and emission emi at 298 nm. The method shows high sensitivity with linearity range from 0.07 to 0.4 5g/mL. The lower limit of detection (LOD) was found to be 1.127 x 10-3 5g/mL, 4.202 x 10-3 5g/mL and limit of quantification (LOQ) was found to be 3.415 x 10-3 5g/mL, 1.273 x 10-2 5g/mL in formulation and biological fluids, respectively. The different experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The proposed method was applied successfully for determination of tramadol in the pharmaceutical preparation and biological fluids. The percentage recovery was found to be 99.477 ± 0.73 and 102.48 ± 1.433 for formulation and biological fluids, respectively.