Microstructure can play a vital role in defining mechanical properties of metallic materials. To elucidate this correlation in the case of Ti–15Mo–3Nb–3Al-0.2Si (TB8) alloy, herein, we designed various microstructures via heat treatment exploring the effects of grain size, precipitates and segregation on crack initiation behavior during tensile tests in metastable β-Ti alloy. After solution treatment at 830 °C, the TB8 alloy with equiaxed β grain displayed a good fracture elongation of 30.2 ± 0.63%. The adiabatic shearing band and β→α phase transformation were activated to increase the compatible deformation capability during tensile testing; however, the phase transformation caused the stress concentration in the boundary, resulting in crack initiation. For the samples prepared using solution and low aging at 440 °C, large grain, elements segregation at grain boundary and incomplete precipitates induced a slight reduction in ultimate tensile strength and elongation. After solution and aging at 520 °C, the short-rod or/and lamellar α phase precipitated in β grain effectively enhancing ultimate tensile strength (1398.71 ± 15.6 MPa). The increased boundaries provided the interface or precipitation strengthening effect, but high-density dislocations were also accumulated at the β/α interface, causing unstable deformation and crack initiation. These findings advance our understanding of the correlation between microstructure and crack initiation, and provide a basis for designing and customizing the mechanical properties of metastable β-Ti alloy.