In this study, corncob acid hydrolysate was used as a substrate for bacterial cellulose (BC) production by Gluconacetobacter xylinus. After 2 weeks' static fermentation, a BC yield of 4 g/L could be obtained. Both effects of medium composition and fermentation condition on the BC production were evaluated. Most extra substrates (carbon and nitrogen sources) except mannitol, butyric acid, and levulinic acid showed no effect on the improvement of BC yield. Fermentation condition including fermentation mode, inoculation concentration, and initial pH showed certain influence on the BC yield and thus should be well controlled. The analysis by field emission scanning electron microscope (FE-SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) showed that the BC sample had obvious nano-network structure, clear functional groups that were found in cellulose, and relatively high crystallinity and crystallinity index value. Moreover, the BC sample had great water-holding capacity. Overall, corncob acid hydrolysate could be one promising substrate for BC production.