To prevent illegal use of clenbuterol and for quality control in the food industry, more efficient and reliable methods for clenbuterol detection are needed. In this study, clenbuterol was detected using a spectral imaging surface plasmon resonance sensor system via two inhibition methods: (1) the target site compensation method, in which anti-clenbuterol antibody was immobilized on the sensor chip as a bioprobe and (2) the solution competition method in which a clenbuterol-BSA conjugate was immobilized on the sensor chip as the bioprobe. The detectable clenbuterol concentration ranged between 6.25 and 100 mu g/mL for both methods. The clenbuterol limit of detection for the target site compensation method and solution competition method are estimated to be 6.7 and 4.5 mu g/mL, respectively. The proposed methods were successfully applied to the detection of clenbuterol molecules and were found to have high specificity and high-throughput and were label free and operationally convenient.