Selective laser melting (SLM), as one of the most popular additive manufacturing (AM) techniques, shows more and more utilization potentialities in today’s aviation industry. In order to ensure safe operation of SLM parts, it is important to have their fatigue lives well-predicted and assessed. In this study, the axial high cycle fatigue tests were carried out systematically for SLM produced Ti6Al4V alloy at room temperature (RT) and 400℃. The effects of defect, direction and temperature on fatigue lives are revealed. Pores located at surface and subsurface of the fatigue specimens are certified to be the main origin of fatigue failure. Through statistic method, the median initial pore (seen as semi-elliptical small crack) size were confirmed. The artificial flaw with the same size was made at the surface of one kind of plate specimen using the focused ion beam (FIB). The small crack growth behavior initiated from the surface artificial flaw of the plate specimen was observed using SEM in-situ fatigue system. Based on the da/dN-ΔK data of small cracks and long cracks of different stress ratios, the da/dN-ΔKeff baseline was obtained according to the Newman crack closure model. From the acquired initial small crack size and the baseline data, the total fatigue lives of SLM produced Ti6Al4V alloy were predicted by FASTRAN program. The prediction curves were evaluated by fatigue experiments. The results showed that the prediction curves agree well with the experiment data of different stress ratios.