Cancer-related γ-glutamyl cyclotransferase (GGCT) specifically converts γ-glutamyl amino acids (γ-Glu-Xaa) into pyroglutamate and the corresponding amino acids (Xaa). Here we report a novel GGCT fluorogenic probe “LISA-101” containing a masked O-acylated fluorophore “resorufin” on the side chain of the P^′₁ amino acid (Xaa). Upon GGCT treatment, the P^′₁ amino acid was liberated and spontaneously released the intact fluorophore. Thus, the fluorescence was regained. LISA-101 will expand the strategies for cancer studies.