The chitosan(CS)/polyurethane(PU) blend nanofibers have been prepared for the first time by electrospinning. Formic acid (FA) and Hexafluoroisopropanol (HFIP) were found to be the co-solvent for electrospinning. The CS/PU blend solutions in various ratios were studied for electrospinning into nanofibers. The diameter and morphology of the fibers were shown by scanning electron microscope (SEM). It was found that the average diameter of the chitosan/PU blend fibers became larger, and the morphology of the fibers became finer with the content of PU increasing. To show the molecular interactions, CS/PU fibers were characterized by Fourier transform infrared spectroscopy (FT-IR). Moreover, the antibaterial activity of blend nanofibers against Escherichia coil (E.coil) was measured via optical density method. The blend nanofibers exhibited satisfying antibacterial activity against E.coil, even the chitosan concentration was only 5wt%. Therefore, the spun nanofibers are expected to be used in the native extracellular matrix for tissue engineering.