Iron(III)-tetrasulfonatophthalocyanine(FeTSPc) has been used as a mimetic enzyme in the determination of hydrogen peroxide with thiamine hydrochloride as a fluorogenic substrate. The determinations were carried out in both acidic and basic environments, with different limits of detection and linear ranges. In acidic condition, the linear calibration graph was obtained from 5.0x10−8mol/L to 8.0x10−6mol/L, with a detection limit of 2.1x10−8mol/L H2O2when Na2HPO4-citric buffer solution (pH 2.8) was used as the reaction medium. It was also found that using one of the three polybasic carboxylic acids such as citric acid, tartaric acid and malonic acid as the catalytic reaction medium can lead to particularly sensitive systems, permitting a detection limit as low as 3.5x10−9mol/L H2O2; whereas in basic reaction medium (Na2CO3-NaHCO3buffer solution, pH = 10.0), the linear range of the calibration graph was from 5.0x10−8mol/L to 2.0x10−6mol/L H2O2with a detection limit of 1.4x10−8mol/L. The applicability of the method to the determination of glucose in human serum was demonstrated by investigating the recovery of the known glucose added to human serum.