The microRNAs (miRNAs) are a kind of single strand small noncoding RNAs(approximately 22nt) with probable roles in the silencing gene expressions. To date, a large number of miRNAs have been identified in several organisms, but the function of the vast majority of these molecules remains to be determined. To study their functions, a library of vectors expressing miRNAs, including more than 170 kinds of human miRNAs were developed. The ability of some miRNA vectors to express miRNAs was validated with Northern blot and dual luciferase assays. The results indicated that these vectors could express pre-miRNAs and mature miRNAs in HEK-293 cells. Furthermore, they could suppress the expression of luciferase tagged with a sequence complementary to corresponding miRNAs. All these data show that these miRNA expression vectors can drive functional miRNA expression, and they can be used to screen functional miRNAs.