Long-term in situ cell membrane-targeted bioimaging is of great significance for studying specific biological processes and functions, but currently developed membrane probes are rarely simultaneously used to image the plasma membrane of animal and plant cells, and these probes lack sufficiently high long-term targeting ability. Herein, we proposed an antipermeability strategy to achieve highly specific and long-term imaging of plasma membranes of both human and plant cells using the steric hindrance effect and restriction-induced emission of AIE-active probes based on an updated membrane model. A certain degree of rigidity of plasma membrane containing a large ratio of rigid cholesterol molecules in the updated membrane model provides a promising opportunity to design antipermeable probes by introducing a rigid steric hindrance group in the probe. The designed antipermeable probes can anchor inside plasma membrane for a long term relying on the combination of the steric hindrance effect and the electrostatic and hydrophobic interactions between the probe and the membrane, as well as light up the membrane via the restriction-induced emission mechanism. The excellent performance in imaging completeness and specificity for both human cells and plant cells clearly shows that these designed probes possess outstanding antipermeability to achieve long-term specific imaging of membrane. These probes also show some advanced features such as ultrafast staining, wash-free merit, favorable biocompatibility, good photostability, and effective resistance to viscosity and pH alteration. This work also provides a valuable design principle for membrane probes of plant cells that the designed probes require a suitable molecular size favoring the penetration of small pores of cell walls.