Donepezil hydrochloride containing polyurethane/hydroxypropyl cellulose (PU/HPC) nanofibers were prepared by the electrospinning for transdermal drug delivery. PU/HPC nanofibers were characterized with SEM, DSC, and Pascal mercury porosimetry. Drug-excipient interaction was studied by ATR-FTIR. In vitro release of PU/HPC nanofiber mat (10:2:1) exhibited Korsmeyer-Peppas release kinetics controlled by the diffusion of drug. In vitro permeation studies across skin resembling synthetic membrane demonstrated the flux of model drug. The in vitro cytotoxicity data obtained via MTT assay indicated that PU/HPC nanofiber mat could be well tolerated by the skin and the components was not irritant for the skin.