The present invention intends to provide an assay system using split luciferase that has a remarkably high detection sensitivity. In an embodiment, binding of mutually binding first and second proteins is detected by preparing a first fusion protein comprising the first protein fused with a peptide having the amino acid sequence of amino acid SEQ ID NO: 1 and a second fusion protein comprising the second protein fused with a peptide having an amino acid sequence selected from the group consisting of amino acid SEQ ID NOS: 2 to 6, and allowing the first fusion protein to bind with the second fusion protein to form a complex, and detecting luminescence emitted from the complex.