The aim of this study was to evaluate the biochemical parameters of the horseradish peroxidase (HRP) immobilized on chitosan nanoparticles (CS) and chitosan/poly (ethylene glycol) (CS/PEG) by the ionotropic gelation technique. The optimum temperature was 50 °C for immobilized HRP in both nanoparticles, while the optimum pH of immobilized HRP were 7.0. The kinetic parameters (Vmax and Km) for both immobilizations were close to free enzyme, with a slight increase in both Km (5.11 and 4.779 mM respectively, compared to 4.499 mM from free HRP) and Vmax (2.329 and 2.475 mM/min compared 1.71 mM/min from free enzyme).