Titanium alloy has excellent comprehensive properties, and it is widely used in clinical applications. Studies show that it is helpful to improve the biocompatibility of titanium alloy implant by introducing specific textures to the surface. A pulsed picosecond laser emitting at 1064 nm was used to process titanium alloy (Ti-6Al-4V), and the biocompatibility of laser textures was verified. Orthogonal experiments were designed, and the surface morphology of the samples was analyzed by using scanning electron microscope, scanning probe microscope and confocal microscope. The relationship between surface morphology of titanium alloy and picosecond laser processing parameters was investigated in order to determine the optimal process parameters. Meanwhile, cell culture and actin cytoskeleton staining were conducted. The experiments show that the picosecond laser surface texturing plays a role in promoting and guiding growth of cells.