The invention provides a method of high-throughput sorting of high expression protein-producing cell, which utilizes linking a protein and a transmembrane domain with a self-processing cleavage site and regulating the secretion of the protein or expression of protein on the cell membrane by adding self-processing cleavage enzyme inhibitor. Then, the high expression cell line can be high-throughput sorted by a detection technique. The invention also provides a recombinant nucleotide sequence and a vector used in the method and a cell sorted by the method.