Chitosan-stabilized Prussian blue nanoparticles (CS/PBNPs) were fabricated by a simple synthetic method and used to develop a novel aptamer-based colorimetric assay for selective determination of dopamine (DA). Scanning electron microscopy (SEM) images exhibited a uniform shape of the CS/PBNPs with an average diameter of 37.0 ± 3.2 nm. The CS/PBNPs exhibited strong peroxidase-like activity that catalyzed the reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). Chitosan was used for stabilization of the PBNPs and fixation of the DA aptamer on the surface of the CS/PBNPs. The catalytic mechanism of the CS/PBNPs was confirmed to involve first the decomposition of H2O2 into a hydroxyl radical (˙OH) and then oxidation of TMB by the ˙OH to produce a blue color. An aptamer-based colorimetric assay was made with the CS/PBNPs to detect DA at concentrations of 0.25-100 μM with a limit of detection (LOD) of 0.16 μM. For comparison, a gold nanoparticle (AuNP)-based apta-sensor detected DA in concentrations of 1-25 μM with a LOD of 0.55 μM. The recovery results of DA concentrations (0.25, 0.5, and 1 μM) from spiked human serum were 92.6%, 102.1%, and 103.9%, verifying the reliability and reproducibility of the CS/PBNP-based apta-sensor for determination of DA level in clinical applications. Moreover, compared to traditional immunoassay, this aptamer-based nanozyme activation/inhibition system needs no washing step, which is very useful to shorten the assay time and maintain high sensitivity.