In order to improve the yield of bacterial cellulose (BC), the fermentation medium of BC-producing strain J2 (Gluconobacter) was optimized, and BC ultra-micro-structure was observed. Initially, Plackett-Burman design was employed to evaluate eight variables which were relevant to BC production. Three statistically significant parameters including yeast extract, ZnSO4, ethanol were selected and other 5 variables were not significant (P > 0.05). The optimized levels of three variables were defined by Box-Behnken design and response surface methodology (RSM). BC ultra-micro-structure was observed by scanning electron microscope (SEM) with cotton cellulose as comparison. The results indicated that the BC yield under the optimum fermentation medium was 11.52 g/100 mL, which was as 1.35 times as that under the original fermentation medium. The SEM photos manifested that bacterial cellulose ribbon, with a diameter less than 0.1 microm, was less than cotton cellulose ribbon. The bacteria inside the cellulose net were eliminated after the NaOH treatment.