Abstract: Nano-Au labelled antibody (having its absorption maximum at the wavelength of 528 nm) was prepared by adsorption of monoclonal antibody of clenbuterol hydrochloride on the surface of nano-gold particles, and was used for spectrophotometric determination of clenbuterol hydrochloride in urine. When clenbuterol hydrochloride (the antigen) was present in the urine sample, immunoagglutination between antigen and the antibody adsorbed on the nano-Au particles occured forming a compound complex with its absorption maximum shifted to 534 nm. Linear relationship between values of absorbance and mass concentration of clenbuterol hydrochloride was obtained in the range within 50 μg·L-1, with detection limit (3S/N) of 0.1 μg·L-1. The proposed method was applied to the analysis of a human urine sample, giving result of content of clenbuterol hydrochloride in consistency with the result found by the GC-MS method given in GB standard.