Abstract: A gold immunochromatography assay (GICA) was studied to establish a rapid assay for detection of Gentamicin (GM) .Colloidal gold marked coupled with monoclonal antibody against GM was jet sprayed onto the glass fiber.Gentamicin-BSA and sheep-anti-rabbit immunoglobulins dispersed on a nitrocellulose (NC) membrane to be the test line (T) and control line (C) .Gold immunochromatographic strips which allowed to specifically detect GM were obtained and its sensitivity, specificity, accuracy and stability were evaluated in this study.The results showed that the sensitivity of rapid assay of Gentamicin was 0.7μg /mL and detection time within 10min.The assay showed good stability and specificity, and no cross-reactivity with other analogs of GM.A good consistency was observed between results of GM determination by this method and ELISA.This method was rapid, easy, sensitive, specific, accurate and stable, so it was very simple and convenient to rapidly detect GM on the spot.