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In the present study, first, saffron petal anthocyanin (SPA) and copper oxide nanoparticles (CuO-NPs) were extracted and synthesized, respectively. Then, multifunctional bio-based films based on carboxymethyl chitosan (CMCS), SPA, and CuO-NPs were fabricated using solvent casting method. The mechanical, thermal, physical, optical, antibacterial, toxicity, and pH sensitivity properties of the prepared films were evaluated. The obtained results demonstrated that simultaneous incorporation of CuO-NPs and SPA improved the mechanical, UV absorption, barrier properties as well as the thermal stability of the films. Moreover, the nanocomposite films exhibited a great antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Fabricated nanocomposite films obviously exhibited color change from pink to green in acidic and alkaline media, respectively …