This paper describes an electrochemical immunoassay for simple, fast and quantitative detection of a urinary hippuric acid which is one of major biological indicator in toluene-exposed humans. The electrochemical system of immunoassay was based on the directly osmium complex conjugated with hippuric acid. With the competition between free hippuric acid (HA) and the osmium-hippuric acid conjugate (Os-HA) to bind with antibody hippuric acid (Anti-HA) coated onto gold nanoparticles, the electrical signals were proportional to urinary hippuric acid (HA) in the range of 0.01-5 mg/mL which is enough range to be used for in-field or point-of-care (POC) diagnosis. The proposed electrochemical method can be extended to the applications to detect a wide range of different small molecules in the field of health care.