<p>Acid phosphatase (ACP) is a critical biomarker for prostate cancer prognosis, but conventional detection methods often lack sensitivity and portability. We report a label-free colorimetric platform for ACP detection that integrates in-situ Pd nanozyme synthesis with smartphone-based analysis. ACP hydrolyzes <span>l</span>-ascorbic acid 2-phosphate sesquimagnesium (AAPS) to generate ascorbic acid (AA), which reduces Pd²⁺ to form palladium nanoparticles (Pd NPs) exhibiting peroxidase-like activity. Polyvinylpyrrolidone (PVP) stabilizes the nanoparticles, ensuring consistent linear response even at ultra-low ACP levels. The in-situ formed Pd NPs catalyze hydrogen peroxide (H₂O₂₎ mediated 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, producing a blue color readout. The catalytic mechanism was validated through steady-state kinetics and radical scavenging studies. The sensor achieves a wide linear range from 0.1 to 10 U/L, a low limit of detection (LOD) of 0.043 U/L, and strong anti-interference performance. Coupled with smartphone-based color analysis, this method enables rapid, portable ACP quantification in human plasma with relative standard deviation below 5%. This work establishes a sensitive point-of-care testing (POCT) strategy for ACP and provides a generalized framework for stabilized in-situ nanozyme synthesis, mechanistic investigation, and portable detection in next-generation biosensor design.</p> Graphical abstract <p></p>