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On the basis of conventional enzyme-linked immunosorbent assay (ELISA) and bienzyme substrate recycle, ELISA-bienzyme colorimetric substrate recycle was developed in the present study. The sensitivity of this method increased 15 times than that of ELISA for the measurement of Tau and increased 55 times for p-Tau. The linear detective rang of this method expanded 3 times higher than that of conventional ELISA for Tau and had the same as ELISA for p-Tau. So, ELISA-bienzyme colorimetric substrate recycle could be used to detect abnormally phosphorylated tau in cerebrospinal fluid.