Abstract: B subunit of Cholera toxin canself-assemble to form a five-membered structure and be fused with nanobody toachieve the purpose of multi-nanobody.The constructed expression vector pET25b(+)-CTB-VHH28 was transformed into Escherichia. coli BL21(DE3)and induced with IPTG for expression. The IPTG concentration,expression temperature and expression time were optimized and the obtained recombinant proteins were subjected to SDS-PAGE. SDS-PAGE assay was used to analyze recombinant protein expression,and the molecular weight of CTB-VHH28 monomer is 30 kDa and that of pentavalentis 150 kDa,which are consistent with predicted molecular weight. By optimizing the concentration of IPTG,the expression temperature and the expression time,the optimal expression conditions was:the concentration of IPTG was 0.05 mmol/L,the induction temperature was 16℃ and the induction time was 10 h. After purification by nickel column,high-purity pentavalent nanobody was successfully obtained and the production yield was 20 mg/L. CTB-VHH28 could specifically bind to OTA by activity measurement and direct ELISA assay. By the activity assay,results showed that the fusion proteins CTB-VHH28 could specifically bind to OTA and the IC₅₀ of indirect competitive inhibition ELISA was 0.38 ng/mL under the condition of 2.5% methanol. Besides,CTB-VHH28 performed excellent thermal stability in a temperature of 25~55℃,and showed little change in competition inhibition rate when methanol concentration was no more than 20% in the reaction system demonstrating a good tolerance toward methanol. In summary,the pentavalent nanobody prepared in this study can be used for OTA detection.