A compact optical biosensor for direct detection of thrombin in human blood plasma (HBP) is reported. This biosensor platform is based on wavelength spectroscopy of diffraction-coupled surface plasmons on a chip with a periodically corrugated gold film that carries an antifouling thin polymer layer consisting of poly[(N-(2-hydroxypropyl)methacrylamide)-co-(carboxybetaine methacrylamide)] (poly(HPMA-co-CBMAA)) brushes. This surface architecture provides superior resistance to nonspecific and irreversible adsorption of abundant compounds in the analyzed HBP samples in comparison to standard surface modifications. The carboxylate groups along the polymer brushes were exploited for the covalent immobilization of aptamer ligands. These ligands were selected to specifically capture the target thrombin analyte from the analyzed HBP sample in a way that does not activate the coagulatory process at the biosensor surface with poly(HPMA-co-CBMAA) brushes. Direct label-free analysis of thrombin in the medically relevant concentration range (1-20 nM) is demonstrated without the need for diluting the HBP samples or using additional steps for signal enhancement. The reported platform constitutes the first step toward a portable and sensitive point-of-care device for direct detection of thrombin in human blood.