In the present study, we used a nucleoside derivative 5-vinyluridine (VrU) for labeling during cell division and for tumor imaging in living mice. We demonstrated that the functional nucleoside bearing a 5-vinyl group is metabolically incorporated into cellular RNA and can be used to image RNA using a Diels-Alder reaction. The reagent allows for simultaneous and clear imaging of DNA and RNA in mammalian cells at single-cell resolution. We extended this approach to observe DNA and RNA behaviors in several basic stages of cell division. We further demonstrated that the derivative can be used for fluorescence imaging of tumor in live mice.