Near-infrared (NIR) fluorescent probes possess a characteristic molecular structure with a highly conjugated polyene or polynuclear system, thus exhibiting a long emission wavelength over 650 nm up to 900 nm. They are of increasing interest owing to their capability of deep tissue penetration as well as low spectral interference with biomolecules in the living system. In this review, we will examine typical molecular structures of NIR fluorophores and then describe their enzymatic reactions with peptidases from the viewpoint of chemical design and reaction mechanism. The targeting enzymes will be covered from aminopeptidase, glutamyltranspeptidase, protease to penicillin G-amidase. Further application to tumor imaging and therapy is also discussed as a theranostic approach.