A biotinylated phenazine compound as a phenosafranin conjugate (Biot-PSF) was synthesized and reported for the first time. The new conjugate was found to be useful as a chemotherapeutic agent with a cancer targeting unit for imaging. We conjugated a molecule recognition moiety with the photosensitive phenosafranin (PSF) to apply the innovative compound in antineoplastic therapy and diagnostics. A new Biot-PSF sensing (bio)probe was established as a biotin-avidin ligand receptor. Thus, Biot-PSF can be an efficient pre-targeting agent for therapeutic and imaging applications. Moreover, the Biot-PSF conjugate exhibits good activity against colorectal carcinoma (HCT116) and adenocarcinoma (COLO 205, SW620) cancer cell lines.