To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC(50)) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products.