Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic foodborne contaminant that seriously threatens food safety and public health. To develop a rapid bioluminescent enzyme immunoassay (BLEIA) for detecting AFB1， this study systematically evaluated the soluble expression， purification， and enzymatic activity of three anti-AFB1 nanobody-nano luciferase fusion proteins (G8-Nluc， Nluc-NB26， and Nluc-NB28). Based on the three fusion proteins， BLEIA assays were established， and Nluc-NB26 was selected for the analysis and validation of cereal samples. The results indicated that Nluc-NB26 exhibited the highest soluble expression level and better stability， followed by Nluc-NB28， while G8-Nluc was largely insoluble. Testing with various surfactants revealed that adding sodium lauroyl sarcosinate improved the solubility of G8-Nluc significantly. The purified fusion proteins all exhibited suitable enzymatic and antigen-binding activities. BLEIA results based on the fusion proteins showed the IC50 values for detecting AFB1 were 4.213， 1.697 and 2.169 ng/mL for the Nluc-NB28-BLEIA， G8-Nluc-BLEIA and Nluc-NB26-BLEIA systems， indicating that G8-Nluc-BLEIA had the highest sensitivity， comparable to Nluc-NB26-BLEIA， while Nluc-NB28-BLEIA had the lowest. Considering the soluble expression level， stability， and detection performance of the fusion proteins， Nluc-NB26-BLEIA was further applied to analyze and validate cereal samples. The results demonstrated that this method achieved average recovery rates of 91.1% to 104.1%， comparable to commercial ELISA kits， but with significantly reduced detection time and reagent cost. These findings offer valuable insights for developing rapid and highly sensitive detection techniques for AFB1.