Small proteins that have a high affinity for cancer cell surface markers can be promising cheap alternatives to antibodies (antibody mimetics). Various types of antibody mimetics have thus been extensively developed. We recently found that a target-binding peptide binds to its target molecule more strongly when it is structurally constrained. To apply this finding to the development of chemically synthesizable small antibody mimetics, we have established an efficient method of creating such proteins, named fluctuation-regulated affinity proteins (FLAPs). To identify desirable scaffolds, first, 13 human proteins (46-104 aa) were selected from the Protein Data Bank. Then, thirteen graft acceptor (GA) sites that efficiently immobilize the grafted peptide structure were identified from six small protein scaffolds using molecular dynamics simulation. To assess the designed antibody mimetics in vitro, human epidermal growth factor receptor 2 (HER2)-binding peptides were selected from the anti-HER2 antibody drugs trastuzumab and pertuzumab by calculating the binding energy, and these were then grafted into the GA sites of scaffolds to create 65 FLAP candidates. The FLAP candidates were expressed in bacteria as fusion proteins with Renilla luciferase (Rluc), and their relative binding affinity to HER2 was easily determined by measuring the Rluc bioluminescence intensity without protein purification. Finally, four out of the 65 showed specific binding to HER2 with a dissociation constant (K_D) of 24-65 nM, and these were used for the detection of HER2-expressing cancer cells. Our design strategy will promote the development of antibody mimetics for the effective treatment of cancers and other diseases.