The effect of Nd:yttrium-aluminum-garnet laser on the microtopography and physicochemical properties of Ti6Al4V alloy are investigated in the view of biomedical applications. The surface roughness and hardness for laser treated samples (LTS) at 140 J cm⁻² were measured 7±0.02 and 825 vickers hardness number, respectively. This superior microhardness value is attributed to grain refinement associated with laser melting and rapid solidification. The electrochemical property, mainly pitting corrosion resistance, has been carried out in Hanks salt balanced physiological solution using standard potentiodynamic polarization testing. A higher corrosion potential of −0.21 V was achieved for LTS. At the optimium treating value of laser fluence (140 J cm⁻²), the energy dispersive x-ray analysis showed about a 30% decrease of vanadium. The contact angle measurements also indicated an improved surface wettability (i.e., hydrophilicity) characteristic at 35°. Finally, the cell culture studies provided a useful tool to investigate the morphology and cell cytotoxicity.