APyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied toALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapidanalysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.