One-step immunoassay detects a target analyte simply by mixing a sample with a reagent solution without any washing steps. Herein, we present a one-step immunoassay that uses a peptide mimicking a target analyte (mimotope). The key idea of this strategy is that the mimotopes are screened from an autodisplayed F_V-antibody library using monoclonal antibodies against target analytes. The monoclonal antibodies are bound to fluorescence-labeled mimotopes, which are quantitatively released into the solution when the target analytes are bound to the monoclonal antibodies. Thus, the target analyte is detected without any washing steps. For the mimotope screening, an F_V-antibody library was exhibited on the outer membrane of E. coli with a diversity of >10⁶ clones/library using autodisplay technology. The targeted clones were screened from the autodisplayed F_V-antibody library using magnetic beads with immobilized monoclonal antibodies against food allergens. The analysis of binding properties of a control strain with mutant F_V -antibodies composed of only CDR1 and CDR2 demonstrated that the CDR3 regions of the screened F_V-antibodies showed binding affinity to food allergens. The CDR3 regions were synthesized into peptides as mimotopes for the corresponding food allergens (mackerel, peanuts, and pig fat). One-step immunoassays for food allergens were demonstrated using mimotopes against mackerel, peanut, and pig fat without any washing steps in solution without immobilization of antibodies to a solid support.