The response of human osteoblast-like osteosarcoma cells (MG63) to surface modification of Ti-6Al-4V implant alloy was investigated by Laser Interference Lithography (LIL). In this work, laser interference lithography was employed to fabricate the microstructures of grooves, dots and dimples onto the surfaces of Ti-6Al-4V samples. Two and three beam LIL systems were developed to carry out the experiments. The laser treatment resulted in the increases of the roughness and the contact angle of water on the implant alloy surfaces. The proliferation of osteoblasts was analyzed by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay for the time periods of 4 hours, 2 days, 3 days, and 6 days. The MTT test results demonstrated that the laser treatment surfaces had a positive impact on the proliferation of osteoblast cells after 24 hours. The alloy surface morphology and the morphological changes of MG63 cells cultured on the laser textured Ti-6Al-4V surface were observed by Scanning Electron Microscope (SEM). The SEM results indicated that the osteoblast cells were aligned on grooved surfaces and they were prolonged with the structures. Enzymatic detachment results showed that the 20 µm grooved structures provided the better cell adhesion to the textured Ti-6Al-4V surfaces.