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In recent years, bacterial cellulose (BC) has been fabricated in tubular shape as scaffold for vascular tissue engineering. However, in order to improve the blood compatibility and regenerative ability of BC, BC nano-fibres should be cross-linked with some materials which can prevent the formation of blood clot. In this work, a novel BC-chitosan (CS)/heparin (Hep) composite was prepared. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier-transformed infrared spectroscopy (FTIR) were used to analyse the obtained samples. It is observed by SEM and TEM that the obtained composites remain the three-dimensional (3D) network and porous structure. The results of XRD reveal that the curve of BC-CS/Hep composite assumes the characteristic absorption peaks of BC, CS and Hep. The FTIR results also confirm the presence of CS and Hep on the surface of BC nano-fibres. In conclusion, BC-CS/Hep composites were obtained by the co-synthesis technique and the cross-linking method, respectively. Furthermore, the MC3T3-E1 cells were seeded on the obtained samples to test the cell compatibility. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results indicated that the BC-CS/Hep composites were suitable for cell proliferation and ingrowth.