Lipopolysaccharide (LPS)is the major valuable antigen used in diagnosis of brucellosis. In this study, to extract LPS from B. abortus strain 99 (reference strain), the hot-phenol method was used .Both chemical and biological characteristics were investigated to ensure quality and quantity of LPS to be involved in further diagnostic studies. Sugars represents about 50% of LPS (0.57 mg/ mg). HPLC analysis of the extracted LPS revealed that its purity was the same as of the commercial LPS of Salmonella Typhimurium. To determine the chemical configuration of S-type LPS, Proton-nuclear magnetic resonance (1H-NMR) was used. The extracted LPS could be cleaved typically to yield a lipid A and polysaccharide moieties (two portions) as main structure of Gram-negative bacteria. A glucosamine sugar residue expanded on a wide range region of NMR spectrum. Considering biological features, Rabbit Pyrogen Test (RPT) of extracted LPS showed low pyrogencity. While anti-complement activity of LPS was dose-dependent, high concentration caused high inhibition of hemolysis. Results emphasized the significance of these methods of extraction and characterization in obtaining valuable antigenic fraction LPS to be used in diagnosis or vaccination purposes.