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As an essential enzyme, alkaline phosphatase (ALP) has attracted considerable attention for its regulating effect to the dephosphorylation process in living organisms. Here, we present a colorimetric and surface enhanced Raman scattering (SERS) dual-readout approach for assaying ALP activity. The major advantage of dual-readout assay is the perfect combination of the merit of colorimetric and SERS, which not only allow rapid preliminary discrimination of ALP activity by the naked eye but also greatly improved the detection sensitivity by SERS. Specifically, 4-mercaptophenylboronic acid modified Ag coated gold nanoparticles (4-MPBA-Au@Ag NPs) are employed as the colorimetric and SERS bifunctional reporting nanoprobes. Upon the presence of ALP, the phosphate group in the ascorbic acid 2-phosphate (AAP) is cleaved to produce ascorbic acid (AA), which acted as boronic acid moieties receptors to control the aggregation of 4-MPBA-Au@Ag NPs. The color of 4-MPBA-Au@Ag NPs solutions had changed from bright orange to light brown to dark gray, simultaneously accompanied by a substantial enhancement of SERS-readouts for the strong Raman hot-spots between the aggregation of 4-MPBA-Au@Ag NPs. A distinguishable change in the color was observed at an ALP activity of 5.0 U/L, meanwhile, SERS-readout sensing method showed a good linear relationship from 0.50 to 10.0 U/L (R = 0.997) with an exciting detection limit of 0.10 U/L (signal-to-noise ratio of 3). In addition, the dual-readout approach developed here was applied for ALP inhibitor evaluation. With the simple, rapid/direct readout yet outstanding sensitivity, we anticipate that this method would greatly promote practical application in ALP-related early-stage diseases diagnosis. [ABSTRACT FROM AUTHOR]