Angiogenin is a potent angiogenic factor that is known to play an important role in tumor angiogenesis. In this paper, we investigate the cellular internalization of angiogenin conjugated with its highly specific aptamer. By using fluorophore-labeled aptamer and confocal laser scanning microscopy, we have developed a novel and simple method by which to visualize the real-time process of angiogenin internalization. Specifically, when aptamer-angiogenin conjugates were added into cell cultures, conjugates could be selectively bound to HUVE cells (human umbilical vein endothelial cells) and MCF-7 cells (human breast cancer cells). Nuclear staining and Z-axis scanning studies demonstrated that the aptamer-angiogenin conjugates were internalized to intracellular organelles, and dynamic confocal imaging studies indicated that the conjugates were quickly internalized. These results provide the first evidence that a fluorophore-labeled aptamer can be used as a fluorescent probe to visualize the spatiotemporal process of protein internalization in real time.