In this work, we demonstrate the development of a highly sensitive and selective surface plasmon resonance (SPR) immunosensor for label-free detection of ractopamine (RCT) as low as 0.12 ppb (ng mL(-1)) direct from pork liver samples. RCT is a kind of the beta-adrenergic agonist with N-alkylphenyl substituent, and the development of high performance sensing methods for RCF is a critical issue because of its vital role in human metabolism. To construct the immunosensor system, the biosensor surface was formed by immobilization of ractopamine-ovalbumin (RCT-OVA) conjugate onto an Au-thiolate self-assembled SPR sensor chip by covalent amide binding, and the detection of RCT was performed based on indirect competitive inhibition principle. Detailed works were carried out focusing on optimizing the conditions that affect immunosensitivity, including immobilization buffer, immunobinding buffer, Rd-OVA concentration, antibody concentration, activation time, regeneration buffer, etc. A preferable way to determine the optimal antibody concentration was performed comparing with that used in most other reported researches. Under the optimal conditions, the biosensor exhibits a linear detection range of 0.28-4.29 ng mL(-1), LOD of 0.12 ng mL(-1) and IC50 of 1.17 ngmL(-1). It improved that the developed SPR immunosensor is more sensitive for detection of RCT against other reported ones. (C) 2011 Elsevier B.V. All rights reserved.