The work aims to clarify the fracture mechanism of laser direct melting deposition (LDMD) Ti-6Al-4V alloy by studying the effect of β grain boundary on the nucleation or propagation behavior of cracks, and provide a theoretical basis for improving the mechanical properties of the alloy. The build was formed on Ti-6Al-4V substrate with LDMD Ti-6Al-4V alloy powder. Samples were taken along the scanning direction of the build. The microstructures of the sample and the real-time changes of the microstructure during in-situ tensile test were studied at room temperature. The effect of β grain boundary on crack initiation, propagation and fracture in tensile test was also investigated, and the fracture mechanism was summarized. The microstructure of the LDMD Ti-6Al-4V sample was composed of coarse columnar β grain that grew parallel to the direction of the build macroscopically. Within the β grain, plenty of lamellar α and α colonies with the same orientation in α plates were observed. Hole defects in a small amount were also identified. The tensile sample by in-situ SEM showed that under the transverse tensile load, the initial deformation of the sample was formed around the hole due to the stress concentration and the crack propagated along the β grain boundary. The β grain boundary hindered the tensile force and resulted in the lower elongation of the sample. During the tensile, the microstructure mainly deformed along phase α surrounding β grain boundary and the hole defects caused more serious deformation due to concentration of stress. There was an angle of 45° between the deformation direction and the tensile direction. The hole defect determines the initial deformation position of the sample, and the β grain boundary determines the direction of crack propagation. Since the direction of the tensile specimen taken is perpendicular to the β grain boundary and the sample has a lower elongation, so β grain boundary plays an important role in mechanical properties and fracture mechanism of sample.