Based on the nature of ketamine hydrochloride being a kind of chemical reaction and lively small molecule,immunology principle of the effects of hapten-carrier was used. Phosphorus trichloride (PCl3) was used as a bridge connecting the ketamine hydrochloride covalent molecules to rabbit serum albumin molecules (RSA). Then, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the coupling and immune BALB/c mice. Finally, the dot enzyme-linked immunosorbent assay (dot ELISA) was applied to detect ketamine antibody response level. Protein electrophoresis analysis showed that ketamine and RSA molecular coupling ratio of 39 complete antigens was prepared successfully, its molecular weight is about 78.8 ku and the mice produced antibodies titer of 1 280 antibodies. The test not only established the serological method for detecting ketamine, also made beneficial effort to develop ketamine addiction vaccine.