The dynamics of DNA and RNA structures in live cells are important for understanding cell behaviors, such as transcription activity, protein expression, cell apoptosis, and hereditary disease, but are challenging to monitor in live organisms in real time. The difficulty is largely due to the lack of photostable imaging probes that can distinguish between DNA and RNA, and more importantly, are capable of crossing multiple membrane barriers ranging from the cell/organelle to the tissue/organ level. We report the discovery of a cationic carbon quantum dot (cQD) probe that emits spectrally distinguishable fluorescence upon binding with double‐stranded DNA and single‐stranded RNA in live cells, thereby enabling real‐time monitoring of DNA and RNA localization and motion. A surprising finding is that the probe can penetrate through various types of biological barriers in vitro and in vivo. Combined with standard and super‐resolution microscopy, photostable cQDs allow time‐lapse imaging of chromatin and nucleoli during cell division and Caenorhabditis elegans (C. elegans) growth. Connect the dots: A cationic carbon quantum dot (cQD) probe emits spectrally distinguishable fluorescence signals upon binding to DNA (green) and RNA (red) in live cells, thereby enabling real‐time imaging of DNA and RNA localization and motion. The probe can penetrate through various types of biological barriers in cells and in vivo for super‐resolution microscopy and time‐lapse imaging of chromatin and nucleoli during cell division and C. elegans growth.