In this study, bacterial cellulose (BC)/ poly(vinyl alcohol)(PVA)/collagen (COL) composite films were prepared by using BC film as base material, PVA and COL as reinforcing materials. And 1-ethyl-3(3-diaminopropy)-carbodiimide hydrochloride (EDC·HCl) was used as cross-linking agent to prepare the cross-linked composite films. Chemical structure, microstructure and properties of these composite films were investigated by FT-IR, SEM, XRD, TG, tensile test and water absorbency. Results show that, PVA and COL molecules are adsorbed and deposited inside of 3D nanofiber network of BC, or coated on BC fibers. The composite films after cross-linked by EDC·HCl, its water absorbency decreases, and thermal degradation stability and tensile strength are enhanced, but elongation at break decreases slightly. Compared with BC film, tensile strength and elongation at break of these composite films have a great improvement no matter cross-linked or not.