A coumarin-based terpyridine–zinc complex (1) was developed for sensing pyrophosphate. In aqueous media (CH3CN–HEPES), complex 1 had been proven to be an effective and selective colorimetric and fluorescent sensor for recognition of pyrophosphate over other structurally similar nucleotides and anions. Among all the tested targets, only addition of pyrophosphate resulted in a color change from brown to light yellow with a fluorescence enhancement of about 27-fold. The terpyridine–Zn2+ complex (1) formed a 2:1 binding mode with pyrophosphate by multi-hydrogen bonds. This complex was successfully applied to fluorescence imaging for pyrophosphate in Hi-5 cells and Caenorhabditis elegans, demonstrating its utility as a fluorescent sensor for detecting pyrophosphate in in vivo imaging.