In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody–alkaline phosphatase (Nb–ALP) fusion protein. The SEB-binding nanobodies were obtained from a na?ve phage-display library and the Nb–ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37–ALP, was 3.12–50.0 ng mL?1 with SC50 = 8.59 ± 0.37 ng mL?1. The limit of detection was 1.44 ng mL?1 according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB–Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class Ⅱ molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb–ALP appears to have the potential to replace chemically labeled probes for the detection of SEB.