Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single-and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and beta-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single-or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single-and multi-amiRNA expression vectors was comparable.