Recently, there has been a lot of interest by using nanobodies (heavy chain-only antibodies produced naturally from theCamelidae) as targeting molecules for molecular imaging, especially for the nuclear medicine imaging. A radiolabeled method that generates a homogeneous product is of utmost importance in radiotracer development for the nuclear medicine imaging. The conventional method for the radiolabeling of nanobodies is non-specifically, which conjugates the radioisotope chelating group to the side chain ɛ-amine group of lysine or sulfhydryl of cysteine of nanobodies, with a shortcoming of produce of the heterogeneous radiotracer. Here we describe a method for the site-specific radioisotope99mTc labeling of nanobodies by transpeptidase Sortase A. The radiolabeling process includes two steps: first step, NH2-GGGGK(HYNIC)-COOH peptide (GGGGK = NH2-Gly-Gly-Gly-Gly-Lys-COOH, HYNIC = 6-hydrazinonicotinyl) was labeled with99mTc to obtain GGGGK-HYNIC-99mTc; second step, Sortase A catalyzes the formation of a new peptide bond between the peptide motif LPETG (NH2-Leu-Pro-Glu-Thr-Gly-COOH) expressed C-terminally on the nanobody and the N-terminal of GGGGK-HYNIC-99mTc. After a simple purification process, homogeneous single-conjugated and stable99mTc-labeled nanobodies were obtained in >50% yield. This approach demonstrates that the Sortase A-mediated conjugation is a valuable strategy for the development of site-specifically99mTc-labeled nanobodies.