This paper presents a proof-of-concept microfluidic aptamer-based sensor for thrombin point-of-care testing using electrochemical impedance spectroscopy. The disposable biosensor is composed of a polydimethylsiloxane (PDMS) channel layer over a glass substrate. The substrate surface has an Au working electrode and a Pt reference/counter electrode. In this study, human thrombin was used as a biomarker for disease diagnosis. An unlabeled aptamer specific to thrombin was immobilized on a working electrode and electrochemical impedance was measured as thrombin was injected into the biosensor. Thrombin was detected by measuring change in impedance. The proposed biochip had a detection range of 0.1–100,000 ng mL−1 for thrombin concentration and a limit of detection of 0.1 ng mL−1. The results indicated that our biochip could be an effective tool for other molecular diagnostic systems.