The invention provides a single cell screening method and an application thereof and in particular provides a single cell screening method. The method comprises the steps of preliminary dispersing, auxiliary dispersing with a microscope, counting and diluting, culture solution preparation and culture. The method has the technical effects that digested and dispersed adherent cells or suspension cells are taken as objects and the concentrations of the cells in culture bottles are adjusted to efficiently screen plenty of single cells; the method is easy to operate, does not have requirements on operational experience of operators, can be used for quickly obtaining plenty of sterile single cells with little variation under sterile conditions and has greater application and popularization values in new drug screening, vaccine preparation and monoclonal antibody preparation.