The present invention relates to a modified biotin-binding protein. The modified biotin-binding protein of the present invention includes an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence having one to several amino acid mutations in the sequence represented by SEQ ID NO: 2, or an amino acid sequence having 80% or more identity to the sequence represented by SEQ ID NO: 2, and having a biotin-binding activity, wherein at least one residue selected from the group consisting of: 1) an arginine residue at position 104 of SEQ ID NO: 2; 2) a lysine residue at position 141 of SEQ ID NO: 2; 3) a lysine residue at position 26 of SEQ ID NO: 2; and 4) a lysine residue at position 73 of SEQ ID NO: 2 is replaced with an acidic amino acid residue or a neutral amino acid residue.