To prepare complete antigen of oxytetracycline (OTC) and obtain mouse polyclonal antiserum with good immunological characteristics and establish immunological detection method of OTC. In this study, carboxyl groups were introduced into OTC molecules by diazotization, and then complete antigens were prepared by coupling with carrier proteins using an improved carbodiimine method. The coupling effect of complete antigen was identified by IR, UV and SDS-PAGE. Then the BLAB/c mice were immunized with complete antigens to obtain antiserum(pAb) and identify the antiserum immunological characteristics. After optimizing the reaction conditions of ELISA, indirect competitive ELISA method of OTC was established. The results showed that complete antigen has good coupling effect, and the titer of antiserum of all four mouse were higher than 112 800, indicating that the serum of all four mouse had inhibitory effect. The antiserum produced by mouse number 2 was the best. The sensitivity (IC₅₀) of the indirect competitive ELISA to assay OTC was 32.92 ng/mL, and the limit of detection (LOD) was 1.3 ng/mL. The addition recovery rate of milk samples was between 84.70%～87.45%. The cross-reaction rates with Chlortetracycline and Tetracyclines were 5.27% and 4.10% respectively, and the cross-reaction rates with other competitors were less than 0.4%. This study successfully synthesized OTC complete antigens and prepared OTC antiserum with good immunological properties, and initially established immunological detection method of it.