In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.