In the present paper, porcine pancreas lipase (PPL) was immobilized to a new version of magnetite via a novel stepwise dithiocarbamate/chitosan-based method in alternation to glutaraldehyde. Magnetic chitosan nanocomposite was post-modified to produce dithiocarbamate moieties on the surface through amine functions. Then, immobilization of lipase was successfully achieved on the surface of magnetically separable Fe3O4@CS/NHCS2H via a post-modification. Each step of immobilization was carefully monitored by characterization and all were successfully proved. Comparison of immobilized enzyme with free enzyme showed that the method of immobilization is efficient.