The effects of laser power, scanning speed, and build directions (BDs) on the microstructural evolution and mechanical properties of laser directed energy deposited (LDEDed) Ti6Al4V alloy were systematically investigated in this study. All the finished LDED specimens were not heat treated and the tensile properties were not influenced by surface roughness. Particularly, the differences in grain size, grain boundary distribution, geometrically necessary dislocation (GND) density, grain orientation spread (GOS), and Schmid factor (SF) of the specimens in different BDs were investigated by scanning electron microscopy, electron backscattering diffraction, transmission electron microscopy, and tensile test. The results showed that the aspect ratio, dilution rate, and deposition angle all tended to increase with increasing laser power and scanning speed. The yield strength and the ultimate tensile strength decreased with the increase of laser power, while the elongation was the opposite. The parent grain reconstruction results indicated that the high-temperature transition phase ((3 phase) of the 0 degrees LDEDed specimen was equiaxial, whereas that of the 90 degrees LDEDed specimen was columnar. The differences in the (3 phase of the specimens in different BDs were directly inherited to the corresponding lowtemperature sub-phase (alpha phase), resulting in finer grain sizes, greater GND density, larger GOS, and smaller SF in the 0 degrees LDEDed specimen, thereby contributing to higher strength and hardness. In contrast, these values were reversed for the 90 degrees LDEDed specimen, resulting in better plasticity and toughness. Finally, the anisotropy mechanism of the microstructures and mechanical properties of the LDEDed Ti6Al4V alloy in different BDs were revealed. This work could provide more systematic and comprehensive guidance in parameter optimization and offer valuable insights into the anisotropy mechanism of the microstructures and mechanical properties of LDEDed Ti6Al4V alloy.