Cyclooxygenase-2 is overexpressed in all stages of cancers and thus serves as an attractive biomarker for early cancer diagnosis. In this study, we report a two-photon probe (SCX) for cyclooxygenase-2 which can reflect the intracellular enzyme level and distinguish between cancerous and normal tissue in colorectal cancer patients. SCX is based on a novel two-photon fluorophore (PBT) possessing phenanthrene donor and benzothiazole acceptor, and indomethacin as a COX-2 targeting unit. The in vitro and cell imaging studies revealed that SCX could distinguish the COX-2 expression levels in cancerous and normal cells. Furthermore, for the first time, normal and cancerous tissues could clearly be distinguished in human biopsy samples by using a COX-2 responsible two-photon probe.