Abstract: objective To prepare a monoclonal antibodies (mAbs) against LPS and O-chain of B.melitensis 16M and establish colloidal gold-immunochromatographic assay (GICA) for diagnosis of Brucellosis. Methods B.melitensis 16M was used to immunize Balb/C mice and the mAbs were prepared using hybridoma technique followed by identification of IgG isotype and its affinity. HiTrap Protein G HP affinity chromatography was employed to purify the antibodies, which were labeled with colloidal gold for establishment of GICA .The best optimization reaction pattern was obtained and installed dipstick, which of specificity, sensitivity, stability and positive samples were tested. Results The hybridoma cells of 2D10、3C6、1E10 were obtained and identified as IgG2a and IgG1 of IgG isotypes with affinity constants (Kaff) ranging from 1×10-8 to 2.6×10-9. 3C6and 2D10 were each optimized coating and labeling antibodies. When labeling IgG were 30μg/ml,the detection result were best, meanwhile,to contrast the polyclonal antibody IgG of B.melitensis 16M and 2D10 were coating antibody and labeling antibodies, two different dipsticks， limit of detection were 5.0×103CFU/ml,and no cross-reacting.The keep time were ≥100d.To contrast fluorescent quantitation PCR,the detection rate was 100% from 80 postive samples. Conclusion The established GICA is rapid and accurate for B.melitensis 16M detection with such potential utility as for instant diagnosis of Brucellosis.