Herein we designed a selective and smartphone-based strategy for visual detection of alkaline phosphatase (ALP) by utilizing the property of amino-functionalized copper (II)-based metal-organic frameworks (NH-Cu-MOFs) with oxidase mimic property and fluorescence property. Surprisingly, the oxidase mimic property of NH-Cu-MOFs can work well at a high pH value 8.0. Thus, a cascade reaction between ALP and NH-Cu-MOFs was realized for the construction of a ratiometric multicolor sensing platform through the controllable catalytic activity of NH-Cu-MOFs by pyrophosphate (PPi) and ALP. The catalytic activity of NH-Cu-MOFs was greatly inhibited because of the binding ability of Cu with PPi. When the ALP was added, the catalytic activity of NH-Cu-MOFs was restored and then further catalyzed the o-phenylenediamine to form the 2, 3-diaminophenazine due to the hydrolysis function of ALP towards PPi into orthophosphates. RGB analysis of the fluorescent sample images was adopted for ALP quantitative analysis. Besides, a hydrogel test kit and mobile app for ALP detection were designed as conceptual products for point-of-care. The LODs of the fluorescence sensing platform was 0.078 mU mL and 0.35 mU mL by solution analysis and hydrogel test kit analysis, respectively. This fluorescent visual method was applied to ALP detection in serum samples with satisfying results, which opened a promising horizon for the diagnosis of other biomarkers in clinical serum samples based on ALP-mediated enzyme-linked immunosorbent assay for the development of biomedicine and clinical diagnosis.