The bacterial cellulose-producing strains were isolated and screened from waste tobacco leaves, and the strains were identified by morphologic observation, physiological and biochemical tests and molecular biological identification. Using waste tobacco extract as carbon and nitrogen source, the mixed-strains fermentation was carried out with screened strain and Saccharomyces cerevisiae (1:1), and the effects of waste tobacco extract addition, mixed-strains inoculum, pH, fermentation temperature and time on the yield of bacterial cellulose were investigated. The results showed that the fermentation process conditions were optimized by single factor, Plackett-Burman and Box-Behnken experiments. The results showed that a bacterial cellulose-producing strain YC1 was selected and identified as Gluconobacter oxydans. The optimal mixed-strains fermentation process conditions were as follows: waste tobacco extract addition 10%, strain YC1 and S. cerevisiae (1:1) inoculum 1.0%, initial pH 5.5, fermentation temperature 30 °C and time 6 d. Under these optimal process conditions, the bacterial cellulose yield was the highest (2.71 g/L), which was 1.29 times higher than that of strain YC1 fermentation alone (1.18 g/L).