In order to promote the comprehensive utilization of tobacco waste, the process of fermentation of tobacco waste to prepare bacterial cellulose (baterial cellulose, BC) was studied. The methods of tobacco waste extracting were compared, and the conditions such as inoculum size, temperature, pH and fermentation time were optimized. The repeated fermentation verification was carried out and the structure and properties of BC prepared by fermentation with traditional HS medium were compared. The results showed that tobacco waste extract can directly produce BC as fermentation raw material without additional carbon source and nitrogen source. The optimal conditions for BC fermentation were inoculum size 6%, initial pH 5, temperature 30 ℃, fermentation 7 d. For different batches of tobacco waste, with the increase of sugar content in raw materials, BC production increased from 1.8 g/L to 2.5 g/L. In comparison, the water absorption performance of BC prepared by tobacco waste fermentation is comparable with the traditional HS medium fermented products, and their morphology and structures are not significantly different. Relying on biotechnology to produce BC by using tobacco waste fermentation provides a new way to expand the utilization value of tobacco resources.