ω-Conotoxins MVIIA (ω-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against ω-conotoxin MVIIA. In this study, an MAb against ω-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a (+), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against ω-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against ω-CTX MVIIA was 7.33×10(9) L/mol, and the cross-reaction test showed that the MAb specifically bound ω-CTX MVIIA. The MAb could be used as a specific antagonist for ω-CTX MVIIA in the physiological study on the CaV channels in the nervous system.