In this study, we developed an integrated microbial co-culture system involving Saccharomyces cerevisiae, Arthrobacter nicotinovorans, and the engineered Escherichia coli for the multi-metabolic conversion of tobacco waste biomass and simultaneous degradation of nicotine. The co-culture system aims to achieve efficient ethanol synthesis, nicotine degradation, and linalool production. A. nicotinovorans exhibited strong nicotine degradation capability, removing nearly all nicotine from 50 g/L tobacco hydrolysate within 96 h, while S. cerevisiae achieved up to 13.8 g/L ethanol production in the presence of 50 g/L tobacco hydrolysate with exogenous glucose. Besides, the engineered E. coli demonstrated the capacity to produce linalool, yielding a titer of 0.65 g/L in 50 g/L tobacco hydrolysate with exogenous glycerol. Co-fermentation of these three strains at a 1:3:1 ratio enabled efficient multi-pathway conversion, achieving 0.17 g/L linalool production, 8.3 g/L ethanol, and near-complete nicotine degradation at 10 g/L tobacco hydrolysate, highlighting the impact of inoculation ratio and substrate concentration on metabolic performance. The co-culture system enabled efficient sugar utilization, effective nicotine detoxification, and the production of valuable chemicals, presenting a sustainable strategy for the comprehensive valorization of tobacco waste.