The present invention belongs to the fields of biochemistry and molecular biology, relates to an αO-superfamily conotoxin peptide, pharmaceutical composition, the method of preparation and the use thereof. The present invention also relates to the propeptide of the conotoxin peptide, nucleic acid construct, expression vector and transformed cell and fused protein thereof. The present invention also relates to a method for blocking acetylcholine receptors, and the use of the conotoxin peptide in making a drug. The αO-superfamily conotoxin peptide of the present invention can block acetylcholine receptors (nAChRs)(e.g. α9α10nAChR), and NMDA receptors(e.g. NR2C NMDAR)specifically, and is active in treating neuralgia and addiction, being used for cancer chemotherapy for treating breast cancer and lung cancer, helping wound healing, and treating epilepsy and ischemia, so it has a good application prospect in the preparation of an analgesic medicine, addictive treatment medicine and neuroscience medicine etc.