Pullulan, an exopolysaccharide produced by a yeast-like fungus Aureobasidium pullulans, has been used for various applications such as a blood plasma substitute, a food additive, an adhesive additive, and a flocculant. The goal of this study was to enhance the pullulan production by optimizing culture medium and pH profile on a batch fermentation with A. pullulans. Various carbon sources (sucrose, glucose, fructose, and lactose) and their concentrations, yeast extract concentration (3, 5, 7.5, 10 g/l), fermentation temperature (25, 28, 30, 32°C), and various pH profiles were examined. The optimal growth condition for pullulan production by A. pullulans has been found as 75 g/l of sucrose as carbon source, 3 g/l of yeast extract, and cultivation temperature at 30°C. Under these conditions with constant pH at 5, the final pullulan concentration was 19.3 g/l with 0.20 g/l/h maximum production rate. A bistage pH profile, which included an initial pH at 2.0 for the first 72 hours of fermentation and then switched to pH 5.0 (constant) until the end of fermentation, was used. The results demonstrated that pullulan production could be enhanced to 23.1 g/l after 7-d cultivation with 0.63 g/l/h maximum production rate.