The trace amounts of tau protein in the blood are considered a significant biomarker in the early diagnosis of Alzheimer's disease. Herein, an electrochemical biosensor has been developed that utilizes a bifunctional nanoenzyme signal amplifier (Nanosa), which incorporates hybridization chain reaction (HCR) amplification and peroxide-mimicking nanoenzymes enhancement to enable ultrasensitive detection of tau. The Nanosa utilizes a peroxide-active MoS2/Au substrate on which DNA aptamer and hairpin probes are fixed as signal amplifiers capable of triggering HCR. The Fc molecules were initially aggregated at the electrode interface through HCR amplification, and were tested by adding an electrolyte solution containing hydrogen peroxide (H2O2). Under the catalysis of a peroxide nanoenzyme, the electrochemical signal of Fc molecules was further enhanced. The bifunctional Nanosa can not only achieve the pre-enrichment of Fc molecules at the electrode interface but also catalyze the enhancement of its electrochemical signal. The well-designed and optimized poly adenine tail not only as an electrode coupling agent but also as an excellent antifouling coating. The ingenious design of the Nanosa-based biosensing strategy enables the ultrasensitive analysis of tau with a broad linear range from 0.1 pg/mL to 100 ng/mL and a lower LOD of 33.4 fg/mL. Meanwhile, the outstanding analytical performance of this Nanosa-based biosensing strategy enables its successful application in determining tau levels in real plasma samples, and showed good agreement with results obtained using a commercial ELISA kit.