Effect of two-stage controlled pH and dissolved oxygen concentration (DO) on pullulan production was investigated. Lower pH and higher DO controlled supported the cell growth of Auerobasidiumpullulans CGMCC7055, while higher pH and lower DO controlled stimulated pullulan synthesis. Maximum pullulan production (80.67g/L) was obtained at initially lower pH (3.0) and higher DO (50%), subsequently higher pH (5.0) and lower DO (30%). And was 17.59% (w/w) and 16.07% (w/w) more than those achieved at a two-stage pH (initially pH 3.0 and subsequently 5.0) with uncontrolled DO, and a two-stage DO (initially 50% and subsequently 30%) with uncontrolled pH (initial pH 6.0). Results indicated that combination of the two-stage controlled pH and DO significantly supported mass production of pullulan.This new and improved pullulan production process methodology would conceivably provide significant contribution and insight, and possibly also to other relevant fermentation processes for the improvement of product yield and productivity at the industrial scale.