Plant cells have a unique organization of microtubules during mitosis and cytokinesis. However, it is difficult to observe cell division in plant cells because of the distortion of images caused by thick samples. We addressed this issue by two-photon excitation coupled with spinning-disk confocal microscopy. Herein, we describe the method for observing microtubules and nuclei during cell division in tobacco BY-2 cells by two-photon spinning-disk confocal microscopy. The protocol includes the transformation of BY-2 cells with a plasmid for the expression of tubulin and histone markers, preparation of cells for microscopy, and operation of the two-photon spinning-disk microscope.