Use of fluorescence spectrophotometry for the determination of procaine hydrochloride in injection solution of procaine hydrochloride in suggested based on fluorescence property of 4-aminobenzoate which is a hydrolysis product of the procaine hydrochloride in pH 12, with exciatation wavelength and emission wavelength at 268 and 340 nm, respectively. Under this condition, a linear relationship is obtained between procaine hydrochloride and the relative fluorescence intensity. The linear regression equation of the calibration graph is c = 0.022I-0.081, with a linear regression correlative coefficient of 0.9996 and relative standard deviation (RSD) of 3.4%. Influences of pH, foreign ions, kanamycin, gentamycin, benylpencillin, streptomycin and standing for time on the determination of procaine hydrochloride have been examined. The reasons that the relative fluorescence intensity of procaine hydrochloride increases with a rise in pH and with standing for time are discussed. The detection limit of this method is 49 ng.mL-1. The results obtained by the method agreed with those by the official method. The method is simple, and available to be used in the determination of procaine hydrochloride in injection solution.