Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P₂ exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P₂-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P₂, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P₂ binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P₂-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P₂. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P₂ probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P₂-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.