Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His(6) encoding recombinant ANEP with a His(6)-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.