Ractopamine (RCT) is banned for use in animals in many countries, and it is urgent to develop efficient methods for specific and sensitive RCT detection. A label-free indirect competitive surface plasmon resonance (SPR) immunosensor was first developed with a primary antibody herein and then improved by a secondary antibody for the detection of RCT residue in swine urine. Meanwhile, a pre-incubation process of RCT and the primary antibody was performed to further improve the sensitivity. With all the key parameters optimized, the improved immunosenor can attain a linear range of 0.3-32 ng/mL and a limit of detection (LOD) of 0.09 ng/mL for RCT detection with high specificity. Furthermore, the improved label-free SPR immunosenor was compared thoroughly with a conventional enzyme-linked immunosorbent assay (ELISA). The SPR immunosensor showed advantages over the ELISA in terms of LOD, reagent consumption, analysis time, experiment automation, and so on. The SPR immunosensor can be used as potential method for real-time monitoring and screening of RCT residue in swine urine or other samples. In addition, the design using antibody pairs for biosensor development can be further referred to for other small molecule detection.