Monoclonal antibodies (mAbs) are a valuable biomaterial for basic life sciences and industrial purposes. The production of the mAb is time and effort intensive. In this report, we established a time- and labor-saving method for the mAb production. Because membrane-type immunoglobulin on a hybridoma cell surface and its secreted form, called as antibody, share the same binding property to the antigen, the fluorescence-labeled antigen bound to membrane-type immunoglobulin can be used as a screening marker. In the method, a hybridoma labeled by a fluorescent antigen was selected and sorted singly into 96-well plate using flow cytometer. Model experiments indicated that the method is highly efficient to obtain good mAbs suitable for Western blotting and immunofluorescence. Notably, most mAbs established by this method belonged to the IgG isotype, which is preferred over the IgM counterpart. Using a high throughput flow cytometer, the method avoids tedious repeated screening and cloning processes. Because the method uses conventional myeloma for cell fusion and all reagents required in this method are commercially available, all research laboratories can apply the method to obtain mAbs efficiently. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.