Gold nanoparticles (AuNPs) are the most commonly used signal materials in lateral flow immunoassay (LFIA). However, the assay sensitivity of traditional AuNP-based LFIA is usually limited by the incomplete competition between free target analytes and immobilized antigens for the binding of AuNP-labeled antibodies. To unfreeze this limitation, here, asymmetric Au-SiO Janus NPs (about 66 nm) were designed and synthesized. Au-SiO Janus NPs can assemble into snowman-like anisotropic structures and combine two different physicochemical properties at their opposite sides, where the AuNP side mainly possesses the antibody conjugating and signal providing functions and the SiO side primarily offers the stable function. In virtue of the unique asymmetric nanostructure, only the AuNP side can interact with target analytes by specific antigen-antibody interactions, which could significantly improve the efficiency of competition. Selecting furazolidone as a model analyte, the immunoassay biosensor showed a limit of detection as low as 0.08 ng/mL, 10-fold decreased than that of the AuNPs-LFIA. Moreover, the Au-SiO Janus NP lateral flow immunoassay was well applied in chicken, pork, honey, and beef food samples with visual detection limits of 0.8 ng/g, 0.16 ng/g, 0.4 ng/mL, and 0.16 ng/g, respectively. The Au-SiO Janus NPs possess the advantages of both materials, which will broaden their applications as a potential alternative in the rapid and sensitive detection of antibiotic residues.