The method for immobilizing antibodies on nanoparticles significantly impacts the analytical performance of lateral flow immunoassay (LFIA). As both Fc-binding peptide (FcBP) and Staphylococcal Protein A (SPA) enable the oriented immobilization of antibodies in a similar manner, do LFIA methods based on these two modes exhibit differences in analytical performance? To address this question, AFB1 antibodies were immobilized on Europium nanoparticles (EuNPs) using non-oriented immobilization, oriented immobilization through SPA and Fc-binding peptide FYEILHGAC, generating the immunoprobes Ab@EuNP, Ab-SPA@EuNP, and Ab-FcBP@EuNP, respectively. The corresponding LFIA methods were developed and the performance of the three methods was compared. The results show that the Ab-FcBP@EuNP method immobilizes more “effective antibodies” compared to Ab-SPA@EuNP and Ab@EuNP. When an equivalent amount of “effective antibodies” is immobilized on EuNP, the IC50 value of the LFIA method based on Ab-FcBP@EuNP is significantly lower (2.58 and 3.61 times) than that of the LFIA methods based on Ab-SPA@EuNP and Ab@EuNP, respectively. The LFIA method utilizing AbFcBP@EuNP displays detection biases for AFB1 spiked in rice and maize matrices ranging from ? 8.53% to 3.90%, and biases in the detection results for certified reference materials (CRMs) of rice and maize containing AFB1 range from ? 11.06% to 6.33%. These results suggest that the LFIA method employing FcBP-based antibody immobilization exhibits superior sensitivity, accuracy, and interference resistance compared to the SPA-based method. The outcomes of this study validate that EuNPs functionalized with FcBP streamline antibody immo bilization, minimize antibody usage, and improve the analytical performance of the LFIA, thus demonstrating favorable potential for practical implementation