This paper, for the first time, presents a wavelength-scanning surface plasmon resonance microscope (WS-SPRM) as a label-free biosensor capable of measuring cell-substrate interaction. The approach utilized a liquid crystal tunable filter (LCTF) as a fast and flexible wavelength-scanning device that can implement a wavelength-scanning and SPR imaging cycle within 1 s. The system was verified by monitoring the dynamics of cellular processes including cell detachment and electroporation of individual cells. It was found that the WS-SPRM presented better performance than the intensity-based SPRM (I-SPRM) in the imaging of cell adhesion. The results also indicated that the WS-SPRM exhibited a larger dynamic range in monitoring cell electroporation than that of I-SPRM. In summary, the developed WS-SPRM in this study provides a promising technique for real-time monitoring of cell-substrate interaction.