Abstract: Objective To facilitate rapid construction of RNAi vector. Methods After inversely inserting the U6 and H1 polymerase Ⅲ promoters into the pBluescriptⅡbackbone vector, only need two short reverse complementary oligo nucleotides, the RNAi vector with one interference cassette could be conveniently constructed. Using two isoaudamers MunⅠ and EcoRⅠwhich could generate compatible sticky ends, several cassettes could be readily fused together to produce the ultimate multiple-site targeting RNAi vector. Using this method, we constructed RNAi vectors targeting green fluorescent protein (GFP) and firefly luciferase (LUC) gene separately or both, in order to test the knock down property of these vectors. Results Constructed single and two site targeting RNAi vectors could get desirable knockdown effects, in which single site targeting vector could get about 90 percent knock down efficiency whereas two site targeting vector could get about 87.5 percent knock down efficiency either. Conclusion Our RNAi vector construction strategy is so efficient and time-saving that it can be used for knocking down several genes simultaneously in same cell.