Mechanobiology is a rapidly advancing field, offering innovative insights and therapeutic strategies for a range of pathophysiological conditions. Central to this progress is the ability to replicate complex cellular and tissue microenvironments in vitro. Microfluidic systems, with their precision-engineered designs, dynamic fluid control, and spatial manipulation, serve as versatile platforms for this purpose. By effectively integrating modeling and sensing capabilities, these systems further provide valuable insights into complex cellular mechanosensing events. In this review, we highlight the latest advances in microfluidic technologies, their significance in replicating living microenvironments, and the challenges they present, with a particular focus on their contributions to unraveling mechanobiological mechanisms and exploring potential therapeutic strategies.