Although fluorescence imaging diagnosis of the differences between cancer cells and normal cells by targeting ligand-based fluorescent probes is useful for recommending personalized therapy to patients, using the differences to diagnose a wide range of cancers is often not possible due to the genetic or phenotypic heterogeneity of cancer cells. In this work a 2-(diphenylphosphino)phenol-functionalized pyronin POP was presented as a dual-channel fluorescence agent for diagnosing a wide range of cancer cell types. The agent could efficiently penetrate cancer cell, rather than normal cell, membranes by active transport of the organic-anion transporting polypeptide (OATP) transporters overexpressed in many types of cancer cell, and is then activated by intracellular cysteine (Cys) and glutathione (GSH) to produce green-emission aminopyronin NP and red-emission thiopyronin SP, thereby enabling its use in dual-channel fluorescence diagnosis of a wide range of cancer cells with excellent contrast. Crucially, POP also displays the ability of dual-channel fluorescence diagnosis of cancer tissues from tumour xenograft models of mice and harvested surgical specimens of patients, thus holding great potential for clinical applications.