Abstract: The flow injection chemiluminescence method for determining alkaline phosphatase (ALP) was based on the catalyzed reaction of cortisol-21-phosphate to produce cortisol which was detected by its chemiluminescence reaction with lucigenin (NN'-dimethyl-9,9'-biacridinium dinitrate). Sample (100 µL) was injected into a Tris hydrochloride buffer carrier stream (0.5 ml/min) at pH 10 which was merged with the reagent stream (1.5 ml/min) containing 0.1 mg/mL lucigenin and 0.06% cetyltrimethylammonium bromide in 0.1 M NaOH. The chemiluminescence was detected using a photomultiplier tube. The calibration graph for 4-50 pM-ALP was presented. The detection limit was 1 pM-ALP and the RSD (n = 5) was <6.1%. The method was applied to FIA for 17-α-hydroxyprogesterone and human chorionic gonadotropin in urine or serum using ALP as the enzyme label. The results correlated with those obtained by other methods.