We attempted to develop a super high-sensitive measurement for determination of a small amount of protein by combination of ELISA and enzyme cycling methods. Because protein cannot be amplified, we must amplify the signal intensity corresponding to the amount of protein. For this purpose, we noted that an enzyme cycling method could amplify the signal released from ELISA. In our measurement system, we adopted the thio-NAD cycling method as one of the enzyme cycling methods following ELISA. For example, we employed alkaline phosphatase as an enzyme labeled with an antibody and androsterone phosphate as a substrate for the alkaline phosphatase in ELISA. We then applied a dehydrogenase with a coenzyme, thio NAD, to the thio-NAD cycling method. The signal was measured as the absorbance of thio-NADH, which is generated by the dehydrogenase from the substrate, androsterone, and the coenzyme, thio-NAD. Our measurement system showed that the detection limit of alkaline phosphatase reached 10(-20) mol and that the detection limit of a given protein was 10(-18) mol. Our measurement system will be a powerful tool for diagnosis and assessment of the progress of a disease.