In this study, we have screened a mutant strain of Aspergillus japonicus-VITSB1 by UV rays and EMS mutagenesis. The resulted mutant strain showed an increased molecular weight, less pigmentation with hyper production of pullulan as compared to parent strain. The various physical parameters were optimized and it was found that incubation temperature 35°C, rpm 200, incubation time 7 days, initial pH 6 and the inoculums size of 10×106 were the optimum condition for the maximum production of pullulan. Considering these optimized parameter, a response surface methodology was employed to understand the interactions among the components of the complex media with second order quadratic model. Results indicated that the concentration of peptone has significant influence in pullulan production as compared with yeast extract. Understanding the effect of complex nitrogen sources in the media using statistical methods has resulted in 73.63 g/L pullulan production which is higher as compared to earlier reports.