It is intended to provide a target substance detection element wherein a target substance capturing body for capturing target substances is immobilized with good orientation in a desired region on the surface of the target substance detection element, a method for producing the target substance detection element, and a detection method using the target substance detection element. The present invention provides a target substance detection element for detecting the presence or absence or concentration of a target substance in a sample, characterized in that: the target substance detection element includes at least a detection substrate including plural layers and a target substance capturing body immobilized on the surface of the detection substrate; the target substance capturing body has at least a first peptide region specifically recognizing a first layer of the plural layers constituting the detection substrate and binding to the first layer and a second peptide region specifically recognizing a second layer different from the first layer of the plural layers and binding to the second layer; and the first layer and the second layer are adjacent to each other.