A method of preparing an epitope for a secondary antibody detected protein tag, comprising: constructing a expressing vector, comprising a nucleotide sequence for encoding the secondary antibody detected protein tag comprising at least one epitope selected from at least one primary antibody which is detected by corresponding secondary antibody; expressing the vector in a host cell; and obtaining the secondary antibody detected protein tag which is expressed in the host cell. Further, a method for constructing a protein molecular weight marker ladder, comprising constructing a plurality of recombinant protein tags with different molecular weights, wherein each the recombinant protein tag comprises at least one epitope of primary antibody. Besides, a secondary antibody detected protein tag comprises at least two peptide sequences of epitopes selected from primary antibodies of at least two different species.