Ti6Al4V alloy samples were prepared by selective laser melting, and the effect of microstructure on mechanical properties was systematically studied by optical microscope, scanning electron microscopy and fracture morphology analysis. Comparing the microstructure characteristics under different parameters, the microstructure features show that columnar β crystal is formed along the deposition direction, and the acicular martensite α′ phase is mainly distributed in the crystal. With the increase of α′ phase content, the plasticity becomes worse. By controlling the process parameters, the acicular martensite α′ decomposes in situ to form α + β phase, which is conducive to improving the plasticity. By controlling the laser power and scanning speed, samples with excellent comprehensive mechanical properties were prepared. The tensile strength reached 1 301 MPa, the yield strength reached 1 102 MPa, and the elongation was 7.94%. Tensile properties can be improved by microstructure regulation of in-situ analysis for titanium alloy manufactured by selective laser melting.