Abstract: The stability of enzyme-labeled antibody plays an important role in enzyme-linked immunosorbent assay(ELISA),but the disadvantages in thermal stability of the conventional antibody-enzyme conjugation has restricted the application of enzyme-labeled antibodies in ELISA. In this study,the shark single domain antibody specific to myofibrillar-binding serine protease(MBSP)fused with alkaline phosphatase was expressed and its affinity and thermal stability were investigated. Results showed that when induced with Isopropyl-beta-D-thiogalactopyranoside(IPTG)at 16 ℃,the fusion protein which was about 63 kDa in molecular weight,which could be expressed in soluble with high affinity and thermal stability. The K_D value of the fusion protein to MBSP could reach 7.80×10^-8 mol/L and the binding activity of fusion protein could be kept well when treated at 58 ℃ even for 180 minutes. All the results implied that the fusion protein could be used as a potential immunodiagnostic agent in ELISA.