A dicyanomethylene-4H-pyran based tumor-targeting and biothiol activatable fluorescent and colorimetric probe, named Biotin-Yellow, has been developed with unprecedented turn-on near-infrared fluorescence properties. Biotin-Yellow is non-toxic and can effectively recognize and discriminate human lung adenocarcinoma cells and urothelial (bladder) carcinoma cells from embryonic kidney cells and embryonic fibroblasts. Biotin-Yellow can also be employed for real-time bioimaging tracking of upregulated biothiol activity in cancer cells. The efficacy and biothiol responsiveness of Biotin-Yellow was further validated in vivo in zebrafish and mice xenografts.