It is of significant importance to understand the influence of defects on the fatigue property of Ti6Al4V alloy prepared by selective laser melting for breaking through the limitation of this material in industrial applications. As the defects cannot be completely avoided, with the help of metallographic microscope, electron backscatter diffraction, X-ray tomography, fatigue testing machine, scanning electron microscope and laser confocal microscope, the microstructure and defect characterizations, high cycle fatigue performance and failure mechanism of the material were studied. The results show that, the microstructure of the alloy exhibits the unique process characteristics of additive manufacturing materials. Specifically, the density of the material is 99.99%, and the overall defect size is less than 60 μm. The fatigue limit of the material is 398 MPa, fatigue cracks are formed in the lack of fusion defects of the fracture specimens and the cycles are less than 106. Most of the effective stress intensity factors at the defects of the fractured samples are distributed above the threshold value of short crack fatigue crack growth, which determines the low cycle life of the material. Finally, the K-T model is also introduced to establish an evaluation method for the safe service of the material.