Human Supervillin isoform 4 (SV4)， a bigger splicing isoform of Supervillin， contains extra coding exons 3， 4 and 5 (E345)， compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton， regulated cell migration and cell survival， its functions are still largely unknown. To broaden our understanding， SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins， which contained SV4 specific domain E345， were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum， and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA， western blot， immunoprecipitation， and immunofluorescence. Taken together， our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function.