Heparin was employed as the stabilizing agent in the synthesis of peroxidase-mimicking Pd nanoparticles. The heparin-capped Pd nanozyme can act as both the signal amplifier and the selective binder of protamine. The most efficient nanozyme with the mean size of 3.5 nm consists of 70.8% metallic Pd0 and 29.2% Pd2+ species. Enzyme kinetic studies show that the Km values are 0.036 mM for 3,3',5,5'-tetramethylbenzidine and 78 mM for H2O2. Protamine shows strong affinity to the heparin-capped Pd nanozyme, and induces an apparent aggregation of the nanoparticles. This results in a significant inhibition of the peroxidase-mimicking activities. Hence, the oxidation of TMB by H2O2 to a blue product with a maximum absorption at 652 nm is suppressed. Based on this finding, a photometric assay is developed for the determination of protamine. The linear response is in the concentration range 0.02 ~ 0.8 μg mL-1, and the limit of detection is 0.014 μg mL-1. This assay presents high selectivity toward other biological substances. Graphical abstract Highly active and selective Pd nanozyme was synthesized through adopting heparin as the capping agent for quantitative determination of protamine.