A recombinant protein and a preparation method and application thereof are provided. The recombinant protein includes a peptide, and the peptide includes an amino acid sequence after substitution of the sequence of SEQ ID NO: 1. The substitution includes at least one selected from the group consisting of the following substitutions: the amino acid at position 182 of SEQ ID NO: 1 is substituted with aspartic acid (Asp); the amino acid at position 268 of SEQ ID NO: 1 is substituted with aspartic acid (Asp); and the amino acid at position 384 of SEQ ID NO: 1 is substituted with tyrosine (Tyr). The recombinant protein is suitable for detecting glycated hemoglobin.