In this study, we constructed a novel and simple yeast surface display system with a single expression vector. The newly established system uses a bidirectional expression vector carrying the AGA1 gene driven by the PGK1 promoter in one direction and the AGA2-expression cassette driven by the TEF1 promoter in the reverse direction, and uses the geneticin, a G418-resistant gene, as the selection marker for transformants. Because all the display elements are put into one expression vector, the new system is much simpler to use, and there is no need for any genetic modification of the host strains; therefore, the new system can be used in wild type as well as laboratory strains of Saccharomyces cerevisiae. The display efficiency of heterologous proteins using the new system has been confirmed by displaying enhanced green fluorescent protein and Eimeria tenella (a chicken protozoan parasite) microneme protein2 (EtMic2) on several S. cerevisiae strains. We also tested the new system with an aga2 mutant strain of S. cerevisiae. The results indicate that the native expressed Aga2 protein has no effect on the display efficiency of heterologous proteins. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:443-450, 2014