The indium tin oxide (ITO) conductive film has been prepared on the flexible poly (ethylene glycol)2,6 naphthalate (PEN) substrate by DC magnetron sputtering deposition method. The effect of the substrate temperature and film thickness parameters on the optical and electric performances of the conductive film has been investigated. The results show that the prepared PEN conductive substrate has a low square resistivity of 35 Ω/□ and a high transmittance in visible region is over 95%. Using the fabricated flexible ITO substrate as an anode, an organic light emitting device consisting of PEN/ITO/NPB/Alq3/Al has been fabricated. Meanwhile, the devices with same structure using different substrates have also been fabricated to compare the discrepancy of the performance.