Methane production from waste activated sludge (WAS) anaerobic digestion is always low due to slow hydrolysis rate and inappropriate ratio of carbon to nitrogen (C/N). In this work, a novel approach, i.e., co-digestion of WAS and tobacco waste (TW) using ozone pretreatment, to greatly enhance the production of methane is reported. Experimental results showed the optimal C/N and ozone dosage for methane production was 24:1 and 90 mg/g suspended solids, and the corresponding methane production was 203.6 mL/g volatile suspended solids, which was 1.3-fold that in mono-WAS digestion. Further investigation showed the co-digestion of WAS and TW was beneficial to the consumptions of protein and cellulose; also, the presence of ozone enhanced the disruption of organic substrates and production of short chain fatty acids, which provided sufficient digestion substrates for methane generation. Analysis of microbial community structure suggested that members of the phyla Bacteroidetes and Firmicutes were the dominant species when ozone pretreatment was applied. The findings obtained in this work might be of great importance for the treatment of WAS and TW.