OBJECTIVE To prepare the monoclonal antibody (mAb) against recombinant human interferon-β1a (rhIFN-β1a). METHODS BALB/c mice were immunized with purified rhIFN-β1a. The spleen lymphocyte cells from the one whose blood specific antibody titer exceeded 1:10 000 were fused with Sp2/0 cells. The fused cells went through HAT selection, serial dilution, and positive selection until stable hybridoma cell lines were obtained. The equilibrium dissociation constant (K(D)) of antibodies was calculated based on ELISA result. The cell line with the best KD value was cultured in a stirred-tank bioreactor with a fed-batch strategy to produce secreted anti-rhIFN-β1a monoclonal antibody. The mAb released into the cell culture supernatant were purified by ultrafiltration and protein-G affinity chromatography. RESULTS Thirteen hybridoma cell lines which stably produced anti-rhIFN-β1a antibody were retrieved from standard hybridoma fusion and selection procedures. One of the 13 cell lines, 1E8 with the KD value of 4.6 × 10⁻⁹ mol/L was cultured and antibody titer in the cell culture supernatant reached 1:5000. The mAb with 90% purity was recovered from the two-steps purification. CONCLUSIONS The anti-rhIFN-β1a mAb with high purity has been successfully obtained.