Mechanobiology is the study of how mechanical forces affect biological systems, including cells and tissues. The two-photon lithography (TPL) as a powerful 3D printing technique allows the creation of 3D complex structures at a microscopic scale. By applying the TPL into the mechanobiology studies, researchers could create precise structures that mimic the mechanical properties of biological system, allowing for the study of mechanobiological processes in a controlled environment. This implies applications in tissue engineering, drug screening, and fundamental research into the mechanisms of mechanobiology. In this review, we highlight recent advances in TPL for mechanobiology studies, as well as the potential future directions for this promising field.