<p> A novel colorimetric aptasensor based on silver nanoparticles (AgNPs) and Ag⁺-dependent urease inhibition has been designed. Sensitive detection of adenosine triphosphate (ATP) and platelet-derived growth factor BB (PDGF-BB) were achieved by employing the competitive assay and sandwich assay, respectively. The method conjugated the aptamer with magnetic beads to construct a magnetic recognition module, and employed poly-C DNA modified AgNPs as the signal amplification module. Target binding to the magnetic recognition module triggers magnetic capture of AgNPs, which were subsequently oxidized to release abundant Ag⁺. These ions efficiently inhibited urease activity, thus blocking urea hydrolysis and preventing pH increase, thereby achieving cascaded signal amplification. The colorimetric mechanism of this method relies on converting the resultant pH change into a color transition of the acid-base indicator phenol red, with the absorbance ratio of A₅₅₉/A₄₃₂ decreasing as target compound’s concentration increases. To validate the generality of this strategy, we designed competitive and sandwich assays for different targets, both achieving satisfactory detection performance. The competitive assay for ATP exhibited a linear range from 1 nM to 10 µM and a detection limit (LOD) of 0.70 nM, while the sandwich assay for PDGF-BB showed a linear range of 1 pM to 10 nM with a LOD of 0.82 pM. The average recoveries in human serum samples were 90.5% to 112.4% and 82.8% to 117.6%, respectively, indicating the effective matrix interference resistance of the developed method. This method provides a novel and versatile approach for designing aptamer-based biosensors, with significant potential for clinical diagnostic applications.</p> Graphical abstract <p></p>