A new structural fragment was synthesized for construction of protein binding-responsive photoluminescent probes. In complex with protein kinases of the PIM family, bisubstrate inhibitors containing benzo[4,5]seleno[3,2-d]pyrimidin-4-one moiety revealed microsecond-lifetime phosphorescence emission after pulse excitation with near-UV radiation. The phosphorescence signal was substantially (more than 50-fold) amplified by a covalently bound fluorescent dye (PromoFluor-555 or PromoFluor-647) whose absorption spectrum well overlapped with the phosphorescence emission spectrum of the selenium-containing heteroaromatic tricycle. The developed organic small-molecule long-lifetime photoluminescence probes possess subnanomolar affinity towards kinases of the PIM family and reveal especially strong emission signal with PIM-2 isozyme. The developed probes have potential to be used for monitoring of activity of PIM kinases for diagnosis of cancer.