Abstract: The present invention relates to an aptamer screening method, and the aptamer screened by the screening method of the present invention binds to a site other than a site where the antibody binds to the target substance to be applicable in various fields such as sandwich-type biosensors and reduce a significant time without requiring a separate pairing selection process. In addition, such an aptamer has excellent stability and sensitivity compared to conventional preparations comprising an antibody, can be mass-produced at low cost in a short time by a chemical synthesis method, and is easily transformed in various ways to increase a binding force. In addition, the immunoassay method using the aptamer screened by the aptamer screening method of the present invention selectively amplifies only the aptamer binding to the target substance in a heterogeneous sandwich structure to detect a relative fluorescence signal, thereby detecting the target substance sensitively and quickly.