Abstract: In order to investigate the effect of different starch raw materials on pullulan biosynthesis by Aureobasidium pullulans, the starch derived from such crops as cassava, corn, potato, sweet potato and wheat was separately used as carbon source for fermentative production of pullulan polysaccharide. The results showed that cassava starch was beneficial to the biosynthesis of pullulan polysaccharide, and the maximum pullulan yield of 23.96 g/L was obtained. Sweet potato starch was not conducive to the biosynthesis of pullulan polysaccharide, and significantly (P<0.05) reduced the yield and molecular weight of pullulan polysaccharide. Moreover, the analysis and comparison of kinetic parameters and physiological indicators involved in batch fermentation of pullulan polysaccharide was carried out. It was found that cassava starch increased the activities of key enzymes for pullulan polysaccharide biosynthesis and intracellular contents of precursor glucose uridine diphosphate, thereby improved the cell biosynthesis capacity and yield of pullulan polysaccharide. However, sweet potato starch increased the activities of pullulan-degrading enzymes and significantly (P<0.05) decreased the molecular weight of pullulan polysaccharide. The cost of carbon source for batch fermentation of pullulan polysaccharide was evaluated. The results showed that the cost of carbon source for the biosynthesis of pullulan polysaccharide from cassava starch was only 56.6% of that from glucose control group. The results presented in this study would provide one feasible technical reference for the cheap and efficient production of pullulan polysaccharide.