To develop new dyes with mitochondria staining capacity, a series of zwitterionic dyes of hybrid coumarin-difluoroboron have been synthesized. The hybrid dyes are conjugated by 7-diethylaminocoumarin, difluoroboron (BF2) β-diketone and electron-donating group. The dyes show deep-red emission (600–680 nm) and high fluorescence quantum yield in organic solvents. The nature of ICT and zwitterion has been fully researched by TD-DFT and ESP calculations. Co-staining with the Mito–Tracker Green shows staining capability to mitochondria in living cells by confocal cell imaging experiments. This work can provide a new strategy for the design of neutral cellular probes for mitochondria staining.