We developed 152T, a biotinylated near-infrared fluorescent probe with high H2S selectivity and mitochondrial targeting. Biotinylation enhances water solubility and tumor retention, while a specific reaction trigger enables distinct H2S recognition. Colocalization (0.892 with MitoTracker) and in vivo imaging in MCF-7 tumors-showing rapid signal onset, peak at 150 min, and tumor-specific fluorescence-support its use for real-time H2S imaging and cancer diagnosis.