The intrinsic microstructures and defects introduced by selective laser melting determine the mechanical and fatigue performance of final shaped parts. Here the grain type, shape and size were characterized using electron backscattered diffraction. The high-resolution synchrotron radiation and industrial microfocus X-ray computed tomography were adopted to identify the porosity and lack of fusion defects in terms of density, morphology and dimension. The larger-sized defects in comparison with α′ grains are more sensitive to the cracking behavior, typically leading to lower fatigue strength and larger life variation. It is found that the fatigue failure tends to originate from larger incomplete fusion defects near the material surface. Then the fatigue strength in presence of defects was evaluated in combination of statistics of extremes and Murakami approach. Finally, an improved Kitagawa-Takahashi diagram was well established based on the concepts of defect-tolerant design and fracture mechanics, including the safe life region and the defect-induced fatigue life in the finite life region.