Abstract: The bovine genomic DNA was extracted from bovine blood, then bovine mammary gland matrix attachment region (BMARs) was cloned using a pair of primers, which were designed based on the related sequences in GenBank through bio-software Primer5.0 and Vector7.0. Upon preliminary analysis with bio-software, BMARs was TA cloned into PMD－18 T vector. By means of adding Kpn2Ⅰand XhoⅠto 5’upstream of sensitive and antisensitive primers respectively, expressing vector BE was constructed after BMAR was cloned into the downstream of the reporter gene in pEGFP-C1. Bovine ear fibroblast cells were transfected by expressing vector BE with Lipofectamine.Compared with control bovine ear fibroblast cells transfected with pEGFP-C1, the effect of cloned BMR was apparent in dispelling position effect and enhancing gene expression.