Electrospinning is a favorable method for the production of Polyacrylonitrile (PAN)-based carbon nanofibers (CNF). The synthesis of carbon fiber includes a heat treatment process to convert polymer nanofibers into CNF. Oxidative stabilization and carbonization are the main steps of the heat treatment process. In this work, the effect of the stabilization process has been reported on chemical and physical properties of Polyacrylonitrile Nanofiber web heat treated with different substrates. The stabilization of electrospun PAN fibers was performed at two temperatures and dwell time at a heating rate of 2 ℃/min. To investigate the effect of stabilization progress, Fourier transforms infrared spectroscopy was used. Scanning Electron microscopy was used to observe the change in the surface morphology of nanofibers with stabilization progress and Elemental analysis was done using Elemental dispersive x-ray spectroscopy. Experimental results showed that the stabilization of PAN Nanofiber on aluminium foil substrate carried out at 290 ℃ showed complete stabilization. It is observed that Nanofiber diameter reduced with exceeding stabilization temperature. An extended study of stabilization on different substrates demonstrated that the surface or thickness of the substrate did not affect the stabilization results, as proven by FTIR Characterization.