A modified biotin-binding protein comprising an amino acid sequence having an identity of not less than 95% to the amino acid sequence of SEQ ID NO: 2 and having a biotin-binding activity and at least one of the replacements selected from the group consisting of replacements 1) to 6): 1) replacement of the serine residue at amino acid position 36 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 2) replacement of the tryptophan residue at amino acid position 80 of SEQ ID NO: 2 with a hydrophilic amino acid residue; 3) replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 4) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the threonine residue at amino acid position 78 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 5) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond; and 6) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue, replacement of the threonine residue at amino acid position 78 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond, and replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond, wherein the amino acid residue that does not form a hydrogen bond is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine and proline, and the hydrophilic amino acid residue is selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid, and, wherein the modified biotin-binding protein satisfies all of the following properties: i) allowing purification using biotin; ii) maintaining a tetramer structure of a protein comprising the amino acid sequence represented by SEQ ID NO: 2; iii) having protease resistance; and iv) showing high expression in a soluble fraction of Escherichia coli. 1) replacement of the serine residue at amino acid position 36 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 2) replacement of the tryptophan residue at amino acid position 80 of SEQ ID NO: 2 with a hydrophilic amino acid residue; 3) replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 4) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the threonine residue at amino acid position 78 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 5) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond; and 6) replacement of the proline residue at amino acid position 46 of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue, replacement of the threonine residue at amino acid position 78 of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond, and replacement of the aspartic acid residue at amino acid position 116 of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond, wherein the amino acid residue that does not form a hydrogen bond is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine and proline, and the hydrophilic amino acid residue is selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid, and, wherein the modified biotin-binding protein satisfies all of the following properties: i) allowing purification using biotin; ii) maintaining a tetramer structure of a protein comprising the amino acid sequence represented by SEQ ID NO: 2; iii) having protease resistance; and iv) showing high expression in a soluble fraction of Escherichia coli. i) allowing purification using biotin; ii) maintaining a tetramer structure of a protein comprising the amino acid sequence represented by SEQ ID NO: 2; iii) having protease resistance; and iv) showing high expression in a soluble fraction of Escherichia coli.