Selective laser melting (SLM) can effectively replace traditional processing methods to prepare parts with arbitrary complex shapes through layer-by-layer accumulation. However, SLM Ti-6Al-4V alloy typically exhibits low ductility and significant mechanical properties anisotropy due to the presence of acicular α′ martensite and columnar prior β grains. Post-heat treatment is frequently used to obtain superior mechanical properties by decomposing acicular α′ martensite into an equilibrium α + β phase. In this study, the microstructure and tensile properties of SLM Ti-6Al-4V alloy before and after various heat treatments were systematically investigated. The microstructure of the as-fabricated Ti-6Al-4V sample was composed of columnar prior β grains and acicular α′ martensite, which led to high strength (~1400 MPa) but low ductility (~5%) as well as significantly tensile anisotropy. The single heat treatment samples with lamellar α + β microstructure exhibited improved elongation to 6.8–13.1% with a sacrifice of strength of 100–200 MPa, while the tensile anisotropy was weakened. A trimodal microstructure was achieved through multi-step high-to-low-temperature (HLT) heat treatment, resulting in an excellent combination of strength (~1090 MPa) and ductility (~17%), while the tensile anisotropy was almost eliminated. The comprehensive mechanical properties of the HLT samples were superior to that of the conventional manufactured Ti-6Al-4V alloy.