The occurrence of shear instability in metallic glasses is profoundly influenced by the spatial perturbation of material properties. Despite the considerable research on the origin of shear transformation zones (STZ), it remains a challenge to understand the behavior from a structural perspective. In this study, extensive molecular simulations (MD) and synchrotron diffraction were employed to characterize the structural features of medium-range order (MRO) defects in Cu64Zr36 metallic glass. These defects manifest as line-like interruptions in the connections between full icosahedral clusters. And it was also observed that the size and content of these defects are closely related to the history of the glass formation process. Moreover, we explored the contribution of these MRO defects, which are comparable in size to the STZ, in the genesis of shear instability. We uncovered that a large strain gradient exists in these regions under external loads, providing a convenient pathway for STZ activation. Notably, the generation of MRO defects are also going on as the STZ evolves. These insightful findings provide valuable insights and offer a promising avenue for the design and development of advanced metallic glass materials with enhanced structural integrity.