IR-780, a novel near-infrared (NIR) fluorescent probe, was synthesized and applied to living cells. The probe exhibited good fluorescent characteristic, and cell experiments showed the probe had high affinity and without apparent cytotoxicity. Fluorescent image experiments in living A549 (Human lung adenocarcinoma epithelial cell line) and L929 (mouse fibroblast cell line) cells, further demonstrated its potential applications in biological systems. The probe effectively prevented the influence of autofluorescence and native cellular species in biological systems. It also exhibited excellent cell membrane permeability, good photostability, and high sensitivity.