We herein describe a simple, sensitive, and label-free strategy for alkaline phosphatase (ALP) activity based on target-promoted exponential strand displacement amplification (eSDA). The developed system utilizes 3'-phosphorylated DNA probes whose phosphate group at the 3'-end blocks the extension by DNA polymerase. Only in the presence of ALP, the phosphate group at the 3'-end is transformed to the hydroxyl group, which enables the extension by DNA polymerase and produces the double-stranded recognition sequence for nicking endonuclease. Consequently, the effective eSDA is executed, generating a large number of duplex products with the high fluorescence signal from SYBR green I. Based on this novel strategy, we determined the ALP activity down to 0.47 U/L with the high selectivity. In addition, the practical applicability of this assaying system was successfully demonstrated by reliably determining the ALP activity in human serum. (C) 2018 Elsevier B.V. All rights reserved.