Based on the analysis of the static mechanical properties of Ti-6Al-4V specimens fabricated by selective laser melting, the effects of surface defects, internal defects and microstructures on the fatigue properties of specimens are investigated under cyclic loading. The stiffness-fitting fatigue cycle method is used to obtain the cycle times of the specimen in each stage of the fatigue cycle. It is found that the fatigue source nucleation stage is the main reason for the difference in fatigue life of the specimen. The surface of directly formed specimens is seriously viscous and forms several fatigue sources, resulting in very short fatigue life. Polishing treatment can reduce the surface roughness, and the annealing treatment can improve the microstructure and enhance the specimen fatigue performance. After polishing treatment, however, internal defects are exposed on the surface. The cycle of defect nucleation fatigue source of different types and different sizes has a large difference, leading to a greater discreteness of fatigue performance.