Abstract: In this study, pullulan fermentation medium was optimized through response surface methodology. The three main factors that played important roles in pullulan production which identified by Plackett-Burman design, were xylose, ( NH) 2SO4and K2HPO4. Subsequently, the central composite design and response surface methodology were applied to further investigate the optimum concentration of the main factors. The results indicated that the optimized concentrations of the main factors were: xylose 55g /L, ( NH) 2SO40.7g /L and K2HPO45.8g /L.With the optimized medium, maximal pullulan obtained in the shake flasks was 16.25g /L, of which increased27.55% compared with the fermentation before optimization.IR spectra of the samples indicated that, the structure of the purified pullulan and the pullulan standard was identical.Pullulan production was increased significantly in this study.It laid a basis for the scale up of pullulan production from cellulosic materials.