Objective: The aim of this study was to obtain highly sensitive and specific mouse polyclonal antiserum of Clonidine hydrochloride (CLO). Methods: Apraclonidine (ACLO), a derivative of CLO, was acted as hapten of clonidine (CLO) to synthesize immune antigen CLO-BSA and envelope antigen CLO-OVA by glutaraldehyde (GA). The coupling effect was identified by ultraviolet scanning and polyacrylamide gel electrophoresis. Then, BALB/c mice were immunized and their immunological characteristics were identified by indirect ELISA and indirect competitive ELISA. Results: The titer of polyclonal antiserum of all mice were higher than 1∶12 800, and the polyclonal antiserum of No.3 mouse has the best sensitivity, the IC₅₀ was 82.75 ng/mL, and the cross reaction rate with several other common clenbuterol was all less than 0.9%. Conclusion: In this study, successfully synthesized the effective artificial antigen of CLO by GA method, and obtained the mouse polyclonal antibody of CLO with high sensitivity and specificity.