A matrix type transdermal patch for delivery of Nebivolol hydrochloride (NEB), is unique as a beta-blocker. Studies were carried out to investigate the effect of permeation enhancers on the in vitro permeation of Nebivolol hydrochloride across rat skin. Films were prepared by using Eudragit RS100 (ERS100) and hydroxy propyl methyl cellulose (HPMC K100M) polymers by incorporating polypropylene glycol (PEG 400) as plasticizer using solvent evaporating method. A total of eight formulations were prepared by using same drug and different polymer ratio and some formulation prepared without (E1-E4)/with (F1-F4) Dimethyl sulphoxides (DMSO) as permeation enhancers in same concentrations. The maximum percent of drug permeation was observed with ERS100 and HPMC K100M (2:8) monolithic system containing 20% Dimethyl sulphoxides (F1). The in vitro release studies revealed that Dimethyl sulphoxides showed better permeation enhancement than without DMSO and the release was sustained up to 48 h and it follows higuchi kinetics. All the films were found to be stable at temperature of 40±2°C and 75±5% relative humidity (RH) as per the international conference on harmonization (ICH) guidelines (Zone IV) for the period of six months with respect to their physical parameters.