As a transmembrane enzyme, ATP synthase plays an important role in energy metabolism of organ tissues, as well as in tumors. In this study we generated a monoclonal antibody, 6G11, to the catalytic subunit of F1-F0 ATP synthase (ATP5B). The SDS-PAGE result demonstrated that the hybridoma clone had a molecular weight of 50 and 27 kDa components that could be the heavy and light chains of the monoclonal antibody, respectively. Chromosome analysis of the hybridoma clone proved that they had 98 to 102 chromosomal numbers that were the sum of the SP2/0 and spleen cells. Western blot assay revealed that the hybridoma clone reacted specifically with the ATP synthase beta subunit, but not with other proteins. In addition, the subclass of the hybridoma clone was identified as IgG1 by capture ELISA. Furthermore, it demonstrated that the antibody retained stability after half a year. These results indicated that the hybridoma clone 6G11 was a monoclonal antibody with significant stability and special reactivity to ATP5B antigen.