Regulating cell growth, extracellular matrix deposition and mineralization of artificial implants are some important parameters that decide the longevity of implants in the body. Picosecond laser-induced hybrid groove structures have been shown to improve these properties of the Ti-6Al-4V bio-alloy. Two hybrid structures containing groove patterns with periodic and non-periodic substructures therein were generated on Ti-6Al-4V by varying the extent of laser pulse overlapping on sample surface. Laser-induced alteration in surface topography, chemical composition and wettability of Ti-6Al-4V resulted in 3-fold increase in the rate of hydroxyapatite growth, 2.5-fold increment in protein adsorption and 2-fold enhancement in cell adhesion in comparison to pristine sample. While the periodic substructure was found to guide cell growth, the nonperiodic sub structure offered homogenous growth leading to higher overall cell density on the substrate surface.