In the present study, CATALASE nanoparticles were prepared by desolvation method using ethanol and glutaraldehyde as desolvating and crosslinking agents respectively. The factors such as the amount of ethanol and glutaraldehyde, stirring rate, and synthesis time were optimized. Properties of the nanoparticles including particle size, morphology, and structural changes were characterized using spectroscopic techniques such as UV-Visible, FT-IR, DLS, and SEM. Also, their kinetic parameters were determined on based the Michaelis-Menten equation. The results of the optimization studies revealed for a phosphate buffer solution (50 mM, pH 7. 0) contain CATALASE (1 mg/ mL), 4-mL absolute ethanol, and glutaraldehyde (12 mg/mL), up to 70% of the enzyme activity remained. In these conditions, the average of the particles was 53. 8 nm. Investigation of the UV-Visible and FT-IR spectra showed that the formation of the nanoparticles caused the structural changes at the tertiary and secondary levels. Comparing the kinetic parameters of the native enzyme and CATALASE nanoparticles showed that the Vmax is the same and that the Km is increased. The physical stability of CATALASE nanoparticles was also investigated. Results showed that the CATALASE nanoparticles lost only 20% of the activity when stored in phosphate buffer solution for 72 h at 4◦ C, whereas native CATALASE lost 55% under the same condition, implying that the CATALASE nanoparticles were more stable than native CATALASE molecules. Therefore, CATALASE nanoparticles offered a great potential to stabilize enzyme molecules for various applications.