Pullulan is a simple linear polysaccharide produced by Aureobasidium pullulans, and has long been applied to various applications from blood plasma substitutes to environmental remediation applications. This review article presents an overview of pullulan’s chemistry, applications, and state-of-the-art advances in enhancing pullulan production through the investigations of genetic regulations, molecular properties, cultivation parameters, bioreactor design, and downstream processing. The genetic regulations are intended to influence the metabolic pathway on pullulan production and its structural composition. Molecular properties of pullulan, such as molecular weight distribution and purity, are crucial for many applications and vary with different fermentation parameters. This paper summarizes the present concepts, methods, and strategies on pullulan production and its applications, and also proposes future prospects on possible new applications through chemical modification as a new biologically active derivative.