The invention discloses a cationization silk fibroin and a preparation method thereof, and belongs to the technical field of medical high-molecular materials. The cationization silk fibroin is obtained through reaction by coupling protamine sulfate mediated by water-soluble 2-imido thiacyclopentane hydrochloride with silk fibroin activated by sulfo-succinimido-4-[N-maleimide methyl]-cyclohexane-1-carboxylate. The cationization silk fibroin disclosed by the invention has the advantages of good biocompatibility and degradability, controllability in surface charge density, effective DNA (Deoxyribonucleic Acid) compression and protection capacity, higher transfection efficiency and uniqueness in cell targeting and cell nucleus positioning function. The cationization silk fibroin disclosed by the invention can form a cationization silk/gene compound with a gene substance through electrostatic action, and the cationization silk/gene compound is a novel biodegradable cationization silk gene transfer vector with cell targeting and a cell nucleus positioning function.