Hemicyanine dyes have a donor-π-acceptor (D-π-A) system that offers potential applications in mitochondrial-targeted analysis. However, there are currently few hemicyanide dyes with good fluorescent properties such as NIR and large Stokes shifts. In this study, a series of hemicyanine dyes were synthesized and evaluated for their fluorescence properties and cytotoxicity as well as mitochondria-targeted imaging. The fluorescent probe HC-7 exhibited near-infrared fluorescence emission (685 nm) with a large Stokes shift (115 nm). It is highly resistant to temperature, pH, and light, has high stability, and shows excellent mitochondrial targeting capabilities. These findings highlight the potential of hemicyanine dyes for improved fluorescence-based mitochondrial imaging and analysis, which facilitate the practical application of hemicyanine dyes in biomedical systems.