Ti-6.0Al-2.5Nb-2.2Zr-1.2Mo alloy (Ti-6321) exhibits excellent corrosion resistance and impact toughness, which can be used as a deep-sea pressure-resistant hull. However, the alloy tends to have high strength but low plasticity, poor impact toughness, and anisotropic mechanical properties by fabricating by direct energy deposition (DED) technology. In order to improve the impact toughness of DEDed Ti-6321 alloy while reducing its anisotropy of mechanical properties, this study manipulates the microstructure and mechanical properties of the alloy through heat treatment. The research finds that with the increase of the single-stage annealing temperature within the (α+β) two-phase region, the width of the α phase increases, the Ultimate Tensile Strength (UTS) remains essentially unchanged, the Yield Strength (YS) decreases, and the impact absorption energy significantly improves. For double annealing heat treatment at two-phase region, temperature increase at the first-stage annealing enlarges the width and decreases the aspect ratio of the primary α phase. Leading to the decrease of the UTS & YS and increase of the impact absorption energy, with a conversely tendency at 1000 °C. The increase in the second-stage annealing temperature raises the size and volume fraction of the secondary α phase, causing the alloy's UTS and YS to initially rise then fall, with a slight increase in impact absorption energy. The anisotropy in the mechanical properties of DEDed Ti-6321 alloy mainly addressed from the different distribution of α phase orientations at different loading directions, and can be eliminated through sufficient β single-phase zone annealing.