Abstract: The culture conditions of Gluconacetobacter xylinus producing bacterial cellulose utilizing corn starch wastewater (adding glucose 20g/L, corn steep liquor 40g/L, ethanol 150mL/L) were investigated through single- factor and orthogonal tests. The suitable culture conditions were as follows:liquid level was 80mL in 250mL triangle bottle, pH4.0, inoculation volume was 9% (V/V) , culture temperature was 28℃ , the the yield of bacterial cellulose reached the peak (4.41g/L) at this time. The bacterial cellulose was verified by FTIR, SEM was used to observe the surface pattern of bacterial cellulose membrane.