B lymphocyte stimulator (BLyS) overexpression is associated with autoimmune diseases such as rheumatoid arthritis and lupus. BLyS antagonists are new effective therapeutic strategies that have been studied extensively. BLyS-binding peptides, BC originated from computer-aided drug design (CADD), 814 selected from the phage display library, as well as the 3-copy of BC (3-BC), were fused with human IgG1 Fc to constitute peptide-Fc fusion proteins, referred as peptibodies. BP-Fc, a peptibody possessing the identical sequence as BC-Fc but a His tag, was also constructed. The biological activities of these peptibodies were assessed by Enzyme-Linked Immuno Sorbent Assay (ELISA). Furthermore, the potential interacting orientations of BP and 814 with BLyS were studied. At 100 μg/ml, BC-Fc, BP-Fc, 814-Fc and 3-BC-Fc could distinctly inhibit 64 %, 50 %, 73 % and 56 % of the interaction of B cell maturation antigen (BCMA) with BLyS respectively. BP-Fc demonstrated 15 % higher binding ratio with BLyS than BC-Fc at 100 μg/ml. However, 814-Fc displayed at least 39 % higher BLyS-binding activity than BP-Fc at different concentrations. The binding capacity of 3-BC-Fc was slightly superior to BC-Fc. In addition, 814 and BP shared the identical domain on the surface of BLyS which involves in binding with BCMA, but owned the detached orientations. The discovery of possible locations of the BLyS-targeted peptides lays the foundation for the development of novel antagonists. Both BP-Fc and 3-BC-Fc fusion proteins could bind to BLyS in a dose-dependent manner and inhibit BLyS biological activity significantly, which might act as candidate agents for autoimmune disease therapy.