Ectopic osteogenesis refers to the occurrence of osteoblasts in soft tissues other than bone tissue and the formation of bone tissue. The ligamentum flavum is an essential connecting structure between adjacent vertebral lamina, which participates in the formation of the vertebral canal's posterior wall and maintains the vertebral body's stability. Ossification of the ligamentum flavum (OLF) is one of the manifestations of systemic ossification of the spinal ligaments and one of the degenerative diseases related to the spine. However, there is a lack of research on the expression and biological function of Piezo1 in ligamentum flavum. Whether Piezo1 participates in the development of OLF is still unclear. The FX-5000C cell or tissue pressure culture and real-time observation and analysis system was applied to stretch ligamentum flavum cells to detect the expression of mechanical stress channel and osteogenic markers after the effect of different stretching durations. The results showed elevated expression of mechanical stress channel Piezo1 and osteogenic markers with the effect of tensile time duration. In conclusion, Piezo1 involves in intracellular osteogenic transformation signal to promote the ossification of ligamentum flavum. An approved explanatory model and further research will be required in the future.