Yeast displaying techniques have been widely used for identifying novel single-chain variable fragments (scFvs) and engineering their binding properties. In this study, we establish a set of vectors for scFv screening and production in the yeast system of Saccharomyces cerevisiae. This suite includes a display vector pYS for screening of recombinant scFv libraries as well as an expression vector pYE for production of scFv candidates in Saccharomyces cerevisiae. The display vector, pYS, give the identification of the HIV-1-specific scFv clones from one scFv display library by fluorescence-activated cell sorting. Subsequently, the expression vector pYE can offer high quality scFvs of interest up to hundreds of microgram scale for bioactivity analysis. As the result, one identified scFv was confirmed to exhibit HIV-1 neutralization activity in a cell line-based pseudovirus assay. The advantage of this system enables the identical post-translation of mammalian scFvs in the same host cells. Therefore, this vector set can be useful for the rapid screening and expression of antibody genes.