In this study, we designed the processing windows for laser powder bed fusion (LPBF) of Ti-6Al-4V (Ti-64) alloy by using central composite design and made a detailed investigation into the influence of processing parameters on the defects. The purpose is to investigate the effect of defects on mechanical properties. It was found that insufficient energy density could lead to the formation of lack of fusion (LOF) defects and produce non-melted powders on the surface, while excessive energy density could lead to cracks that were detrimental to mechanical performance. In addition, the microstructural evaluation found that relatively low energy density could lead to shorter columnar prior-β grains, while prior-β grains in the sample processed by the high energy density extended almost the entire height of the cross-section, which could lead to the strong mechanical property anisotropy. The prior β grains are formed by heterogeneous nucleation on the partially melted material powder. As the energy input increases, all the powder powders in the molten pool can be melted so that these particles do not act as nucleation sites and the prior β grain can grow through more layers without forming new grains being able to nucleate. The prior β-grain in as-built Ti-64 samples consisted of acicular α’ martensite with myriads of lattice distortions, as a precursor to a phase transition, which lead to strong tensile strength and poor ductility. Annealing heat treatment promoted the improvement of the ductile performance of LPBF Ti-64. Overall, this study provides comprehensive views on the effects of processing parameters (laser power, scanning speed, and hatch distance) on the internal (pores and LOF) and external (unmelted powder, sintering neck, and crack), defects, microstructure, and tensile property evaluation of LPBF Ti-64, which offer insights for the development of additive manufactured titanium alloys with excellent mechanical property.