Ti-6Al-2Mo-2Sn-2Zr-2Cr-2V titanium alloy is prepared using a coaxial powder feeding laser deposition manufacturing process. The microstructures of the alloy in deposited and heat-treated state are studied, and the fatigue properties of the heat-treated alloy are evaluated after heat treatment. The evolution law of microstructure before and after heat treatment is analyzed, and the effects of microstructure and defects on fatigue properties are discussed. Results show that the microstructure of the deposited alloy is composed of thick primary β-columnar crystals, and the interior of the grain is composed of thin lamella α and intergranular β-phases. The volume fraction of the α-phase is significantly more than that of the β-phase. After solution and aging in the (α+β) phase region, the microstructure is still composed of coarse primary columnar crystals, the intracrystalline α-phase coarsens, and the β-phase volume fraction evidently increases. Compared with Ti-6Al-4V alloy samples prepared with the same process, the fatigue performance of the heat-treated experimental alloys is higher in the high-stress zone but lower in the low-stress zone. The difference in alloy element content, phase composition, and microstructure were the main factors affecting the fatigue performance of the deposited and heat-treated alloys. Fracture analysis shows that most of the nucleation sites of the fatigue source are located at the strip-shaped non-fusion defects and the porosity defects, and the larger the diameter of the defects, the closer to the surface, the more obvious the stress concentration, and the lower the fatigue life.