AbstractA highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O 2−) and hydrogen peroxide (H2O2) in the presence of 1‐methoxy‐5‐methylphenazinium methyl sulphate (1‐MPMS) as electron mediator. The produced O 2−and H2O2can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m‐POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10−9mol/I to 10−5mol/I. This chemiluminescent reaction has been coupled to the assay of glucose‐6‐phosphate dehydrogenase (G6PDH), β‐D‐galactosidase (β‐Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, β‐Gal and ALP were 10−18mol, 10−20mol and 10−18mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17α‐hydroxy‐progesterone and DNA h