In the present work, Titanium dioxide (TiO2) micro–nanostructured thin films are deposited by a cold atmospheric plasma jet on carbon fiber substrates. The surface morphology, grain size, and structure phase of TiO2 thin films are investigated by scanning electron microscopic (SEM), X-ray diffraction (XRD), and Raman spectrum. As the discharge voltage increased from 5 to 15 kV, the size of these TiO2 particles decreased from 2 to 3 μm to less than 1 μm. The XRD and Raman spectroscopic results show TiO2 on the carbon fiber surface prepared by atmospheric plasma jet is at the mixture phase of anatase and rutile. We also investigated the adhesion and proliferation assays of MC3T3-E1 preosteoblasts on the samples. The surface with smaller TiO2 particles deposited on carbon fiber is more appropriate for attachment of preosteoblasts. Furthermore, the highest proliferation of MC3T3-E1 was found on a sample with smaller TiO2 particles after incubation. Our data suggest that the increased roughness fosters cell attachment and proliferation on the surface of TiO2/carbon fibers.