On the basis of conventional enzyme-linked immunosorbent assay (ELISA) and dienzyme substrate recycle, An highly specific and ultrasensitive biochemical assay, named ELISA-dienzyme substrate recycle was developed. The detective range of conventional ELISA for measuring recombinant tau and abnormally phosphorylated tau purified from Alzheimer disease brain was 1ng to 32 ng. It was 0.75pg to 200pg and 0.5pg to 50pg by ELISA-dienzyme substrate recycle. Compared with the conventional method, the sensitivity of the technique established in the present study increased 400 and 1300 times, with a enlarged detective range of 8.5 and 10 times, respectively. It could detect abnormally phosphorylated tau in cerebrospinal fluid of Alzheimer disease and used effectively for the diagnosis of the disease.