Objective A novel technology of microcantilever sensor platform for label-free detection of clenbuterol antigen-antibody specificity binding is reported. Methods Thiolated clenbuterol antibody was immobilized on the gold-coated side of the microcantilever by self-assembled monolayer technique. Consequently the deflections of the microcantilever corresponding to the process of injecting different concentration of clenbuterol antigen were real-time monitored by optical lever technique. The activity of thiolated clenbuterol antibody and the clenbuterol antigen-antibody binding on the gold-coated side of the microcantilever were confirmed by an Enzyme-Linked Immunosorbent Assay (ELISA). Results The sensitivity of detecting clenbuterol is better than 1ng/ml. Microcantilevers functionalized with thiolated clenbuterol antibodies were exposed to 1 μg /ml of Chloramphenicol (CAP), and no significant response was detected, which confirmed the specificity of the immobilization method. In addition, clenbuterol antigen-antibody binding on the microcantilever generates a compressive surface stress and the surface stress is linear to logarithm of clenbuterol concentration over a range of concentration from 1ng/ml to 1μg/ml. Conclusions It is possible for detection of clenbuterol by microcantilever sensor platform.