Chitosan, chitosan/45S5 bioactive glass (mBG), chitosan/nanobioglass (nBG), and chitosan/mBG/nBG coatings were deposited on grit-blasted and mirror-polished Ti6Al4 V alloy substrates by electrophoretic deposition (EPD). This study discusses how the surface characteristics of the substrates influence the 3D surface topography, areal surface roughness, morphology, and wettability of chitosan-based thin composite coatings while focusing on tuning the surface characteristics by grit-blasting. The surfaces of coated and uncoated specimens were characterised by SEM analysis with EDX, image analysis, contact angle measurements, 3D surface topography analysis, FTIR analysis, and in-vitro bioactivity studies. The coatings deposited on grit-blasted substrates presented rougher surface topography, higher areal surface roughness values and wettability compared to the mirror-polished surfaces. A deposition mechanism was suggested to clarify the deposition of chitosan/mBG coatings on grit-blasted substrates. Apatite crystals were formed on chitosan/mBG coatings after immersion in SBF for three days. A robust image analysis method was applied to reveal the distribution and area coverage percent of deposited mBG, which demonstrated the increase of mBG coverage with the increases in voltage. The present study shows that grit blasting could be a favourable surface treatment to tailor the surface characteristics of organic-inorganic composite coatings by influencing their surface topography, roughness, and wettability.