An improved peptide library preparation method is described for constructing complete virtual peptide libraries such as a complete virtual tripeptide library, tetrapeptide library, pentapeptide library, hexapeptide library, heptapeptide library, or a complete octapeptide library, etc. The method includes constructing an expression vector for the expression of cyclic peptides. Each cyclic peptide displays an array of peptides of different sizes and sequences, and the number of cyclic peptides needed for constructing a complete virtual peptide library can be dramatically reduced compared with conventional chemical peptide synthesis. Furthermore, the cyclic peptide libraries can be readily reproduced by the expression and purification of the cyclic peptides using the constructed gene libraries. The improved peptide library preparation method can particularly be used, for example, to construct a complete virtual tetrapeptide library, a complete virtual pentapeptide library, a complete virtual hexapeptide library, a complete virtual heptapeptide library, and so on. The improved peptide library preparation method can also be used, for example, to construct a partial pentapeptide library, a partial hexapeptide library, a partial heptapeptide library, and so on. Other related methods and the related expression vectors are also described.