A spectrofluorometric method for the determination of ascorbic acid (AA) based on its activation on the hemoglobin-catalyzed reaction was proposed. The fluorescence intensity of the product was measured under the optimal experimental conditions, i.e. 4.0 × 10^-6 M H₂O₂, 6.0 × 10^-5 M p-cresol, 1.2 M NH₃-NH₄Cl (pH 10.4) and 2.0 × 10^-7 M hemoglobin. The order of additions of the reagents was also studied. The activation of AA was found to be associated with a high ammonia concentration. The linear range of the method was 9.0 × 10^-10 - 3.6 × 10^-8 M of AA. The detection limit was calculated to be 3.0 × 10^-10 M. The relative standard deviation of this method is 1.6% at 7.0 × 10^-9 M for 11 determinations.