Chitosan stabilized iron oxide magnetic nanoparticles (Chit-MNPs) were prepared with the theoretical Chit/Fe3O4 weight ratios equal to 0.5, 1, 2, 3 w/w and characterized by different techniques. The effect of various Chit/Fe3O4 weight ratios on the samples’ hydrodynamic diameter, zeta potential, and isoelectric point was studied by the dynamic light-scattering method. Using TEM analysis, the average magnetic core size was found to be ranging from 6 nm to 9 nm. The obtained results are comparable to those from magnetic measurements. The magnetic measurements confirmed successful chitosan coating on MNPs by means of the decrease in saturation magnetizations from 69.2 emu·g−1 for unmodified MNPs to 66.6, 65.9, 65.5 and 65.2 emu·g−1 for Chit-MNPs at different ratios of Chit/MNPs 0.5, 1, 2, and 3. TGA was used to quantify the amount of chitosan adsorbed on MNPs surface – the max. amount was 0.045 mg chitosan/mg MNPs. The peroxidase-like catalytic activity of unmodified MNPs and chitosan Chit-MNPs in present of a substrate such as N,N-diethyl-p-phenylenediamine sulfate salt (DPD) and hydrogen peroxide was studied. The results confirmed the peroxidase-like activity of samples, – they catalytically oxidise substrate DPD by H2O2 to produce a typical colour reaction. The chitosan coating was found to slightly decrease catalytic activity compared to MNPs at pH 7.4. Despite the fact, these findings can be useful for creating sets of MNPs and Chit-MNPs samples possessing predefined peroxidase-like activity and could be applied for chromogenic reactions in the future.