Amorphous carbon (a-C), one of the most studied carbon-based films, has been widely applied in the filed of artificial joints due to its excellent mechanical and tribological properties and high corrosion resistance. However, low adhesive strength and poor fatigue resistance are fatal shortcomings of this kind of films. In this study, carbon (sp2-C:Ti/sp3-C:Ti) nano-multilayer films (MFs) with sequential sp3-rich and sp2-rich layers were deposited on Ti6Al4V and c-Si substrate by DC magnetron sputtering. X-ray diffraction, scanning electron microscopy, the X-ray photoelectron spectroscopy and the transmission electron microscopy were used to characterize the composition, structure and morphologies of the MFs. In the sp2-C:Ti/sp3-C:Ti MFs, the sp3-C rich layer and the sp2-C rich layer exhibit amorphous features and onion-like structure, respectively. Moreover, the layered structure is continuous in a relatively clear region but with a rough layer interface. The mechanical and the scratch behaviors were elaborately characterized by nanoindentation and scratch tests. The results revealed that the sp2-C:Ti/sp3-C:Ti MFs possess hardness of 21.9 GPa and excellent scratch behavior at sputtering bias voltage −220 V, which are significantly better than single-layer films. Furthermore, the adhesion and tribological behaviors of the sp2-C:Ti/sp3-C:Ti MFs were obtained by Rockwell and ball-on-disk tribometer tests. It was found that the sp2-C:Ti/sp3-C:Ti MFs synthesized under a sputtering bias of −220 V not only exhibit relatively low coefficient of friction (only 0.11) in humid air, but also present lower coefficient of friction (0.085) and wear rate (7.5 × 10−17m3N−1 m−1) of Si3N4 balls in Fetal bovine serum (FBS) solution. In conclusion, the critical loads and wear resistance of the sp2-C:Ti/sp3-C:Ti-MFs were effected by the sputtering bias and the mechanical properties of the MFs. And when sputtering bias is −220 V, the sp2-C:Ti/sp3-C:Ti MFs exhibited the improved mechanical properties, the lowest coefficient of friction (CoF) and the highest wear resistance.