Abstract: Exploiting and utilizing green biomass materials can reduce the consumption of petroleum-based polymers. However, compared with the single bacterial cellulose (BC), BC film always exhibits the poor mechanical properties, which limits its application. In this study, in order to improve the strength and toughness of BC composite films synergistically, BC was treated by alkaline and 2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPO) oxidation to obtain TEMPO-oxidized BC (TOBC), which was utilized as matrix to prepare the TOBC-based composite films enhanced by carboxylic multi-walled carbon nanotubes (CNT) via vacuum filtrating technique. The effect of CNT amounts on the mechanical properties and microstructure of TOBC-based composite films were investigated emphatically and the strengthening and toughening mechanism was also discussed. The results show that: When CNT content is 7.5wt%, CNT-TOBC composite films exhibit the best mechanical properties. The tensile stress, elongation and toughness of CNT-TOBC-7.5wt% composite films are 174 MPa, 10.83% and 12.01 MJ·m⁻³, respectively, which are increased by 56.76%, 144.47% and 295.07% compared with the pure TOBC films, respectively. The improvement is attributed to the hydrogen bonding interaction between CNT and TOBC, the high strength of CNT, as well as the external toughening mechanism. This study provides a feasible method to improve the interface bonding and mechanical properties of composites, and further broadens the application of TOBC in flexible electronic substrates, intelligent packaging and other fields.