A highly sensitive SPR immunosensor has been fabricated by covalent binding of a protein conjugate of 2,4-D (2,4-DBSA) on SAM of 3-mercaptopropionic acid. With indirect inhibition immunoassay, it demonstrated a low detection limit of 0.1 ppb 2,4-D. Taking advantage of the combination of the SAM technique and immobilization of 2,4-D-BSA at controlled surface coverage, a remarkable enhancement of the low detection limit by 10-fold to 10 ppt with no use of any labels was demonstrated, while keeping the response time constant at 4 min.