Copper plays important roles in a variety of fundamental physiological processes. At the cell organelle level, aberrant copper homeostasis in lysosomes can lead to various serious diseases. Herein, a bifluorophore-based, lysosome-targetable Cu2+-selective ratiometric fluorescent probe (V) has been synthesized by reasonable design. The probe V shows high selectivity toward Cu2+ ions over other cations and exhibits high sensitivity (1.45 nM) for the detection of Cu2+ ions. Meanwhile, the probe is cell permeable and suitable for ratiometric visualization of lysosomal Cu2+ in the living cell.