Pronounced changes of acid phosphatase (ACP) in the human body may occur because of several diseases. Unusual ACP expressions might be diagnostically useful as serological and histological biomarkers for specific conditions and for investigations into the pathophysiology of their associated diseases. Recently, artificial enzyme-based colorimetric detection has garnered increased research interest because of its practicability, cost-effectiveness, and flexibility in use. Herein, for the first time, a label-free sensitive visual detection of ACP is proposed based on a signal transduction strategy in conjunction with an enzyme cascade reaction and the nanozymatic activity of the in situ synthesized gold nanoparticles (Au NPs). The ACP-catalyzed hydrolysis of L-ascorbic acid 2-phosphate sesquimagnesium (AAPS) generates ascorbic acid (AA), which in turn reacts with HAuCl 4 to generate Au NPs. The nonenzymatic activity of the synthesized Au NPs toward 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2 O 2 produces a visual readout color that may be utilized to effectively determine ACP concentrations in biological media. The terephthalic acid test confirmed that the size of the Au NPs played a significant role in the nonenzymatic reaction. The quantitative sensing of ACP activity over a wide linear range (0.01−50 mU mL −1 ) with a detection limit of 0.03 mU mL −1 was realized, which endowed the proposed method with high sensitivity for potential real-life applications. Moreover, the present detection approach demonstrated higher sensitivity than a commercially available ACP kit. The approach reported in this study opens a prospect for label-free amplified colorimetric assays of enzymes and biomarkers.