We generated a novel G-quadruplex (G4)-specific endonuclease by fusing a G4 recognition domain of the RHAU helicase with a cleavage domain of the Fok1 nuclease. The fusion protein can specifically bind a parallel G4 and cleave a double-stranded DNA (dsDNA) next to it. The new endonuclease could be used to detect a G4 in a long dsDNA, providing a useful tool for mapping the formation of G4s in the genome.