Functionalized Fe3O4 nanoparticles decorated with carboxymethylated chitosan were developed and used as a novel magnetic support for the covalent conjugation of papain, one of the most important industrial proteases. The analyses of transmission electron micrographs (TEM) and X-ray diffraction (XRD) showed that the size and structure of functionalized Fe3O4 nanoparticles had no significant changes after conjugation with papain. Magnetic measurement revealed that the resultant papain-conjugated nanoparticles were superparamagnetic with a saturation magnetization of 59.3 emu/g. Analyses of Fourier transform infrared (FTIR) spectroscopy and measurement of zeta potentials confirmed the conjugation of papain with the functionalized Fe3O4 nanoparticles. Compared with the native papain, the conjugated papain was found to exhibit enhanced enzyme activity, better tolerance to the variations of medium pH and temperature, and improved storage stability as well as good reusability. Considering that the magnetic separation technique possesses the advantages of rapidity, high efficiency, cost-effectiveness, and lack of negative effect on biological activity, such a bioconjugate system may hold potential applications in food, pharmaceutical, leather, cosmetic, and textile industries.