The present work investigated the characterization of cassava films as a separator/ion-exchange potential in Zinc-air fuel cell (ZAFC). Fuel cell system has promising as a new potential candidate in generating electricity by producing a clean energy conversion device due to their low gas emission. However, not all the organic polymer acts as separator/ion-exchange potential in fuel cell is a biodegradable polymer. The effect of plasticizers on morphologies, structural chain, chemical and physical properties of cassava starch film has been investigated. Mixtures of cassava starch and distilled water with combination of glycerol, sorbitol, sucrose and aliquat (0.5 wt %, 1.0 wt %, 1.5 wt %) as plasticizers was well heated at gelatinized by heating to temperature 70 - 80 oC. Homogenous starch solution was cast onto acrylic plate and allowed to dry in room temperature (25 oC). Dried films plasticized show under scanning electron microscope (SEM) and compound microscope as smooth film surfaces were observed in mixture of glycerol, sorbitol and sucrose due to the fact that glycerol and sucrose are more hydrophilic than sorbitol based on observation of chemical reactions observed by fourier transform infrared spectroscopy (FTIR). As a results, film with 1.5 wt % of aliquat indicates as optimum composition for film transport mechanism, while film with presence of sorbitol, sucrose and glycerol as plasticizers shows more compact and homogenous surface, improves the stability and flexibility on cassava starch films.