Notch signaling mediates intercellular communication required for cell fate specification. Because the majority of Notch proteins exist as an intracellular pool, information on intracellular trafficking of Notch is essential for understanding its regulation. To observe trafficking of Notch protein in vivo, we constructed a Notch protein with green fluorescent protein (GFP) inserted into the intracellular domain. This construct rescued the neurogenic phenotype of Notch mutant embryos and caused the typical Notch gain-of-function phenotype when over-expressed in the wing imaginal disc. Using this and similarly constructed Notch-mCherry fusion, we showed rapid spatio-temporal dynamics of Notch in endosomes. Kinetic analysis revealed that activation of Notch-GFP by ectodomain shedding causes fast and unrestricted nuclear translocation of Notch intracellular domain. During asymmetric cell division of sensory organ precursor cells, distribution of Notch-GFP showed transient asymmetry at the interface of daughter cells, confirming a possible site of regulation that biases Notch signaling level among daughter cells.