Introduction Lipopolysaccharide (LPS), or endotoxin, a major component in the outer cell membrane of Gram-negative bacteria is a very powerful and toxic inflammatory stimulator, resulting in sepsis or septic shock, a significant medical problem affecting about 700000 patients and causing 250000 casualties annually in the United States itself. The detection of LPS is highly importance. However, the currently used enzymatic limulus amebocyte lysate assay is highly susceptible to changes in temperature and pH, interference factors, and requires cumbersome sample preparation. A more cost-effective, sensitive and robust detection method is needed. Objective To design and develop biosensor for LPS detection by assembling a LPS-binding peptide (as LPS receptor) with graphene oxide (GO, as fluorescence quencher). Methods GO was synthesized using a modified Hummer's method. A synthetic LPS-binding peptide was designed, fluorescent labelled, and assembled with GO in PBS buffer solution. The fluorescence recovery of the peptide-GO was measured upon addition of LPS from Gram negative bacteria: E. coli, K. pneumoniae, Samonella Thyphosa, P. aeruginosa, as well as living pathogenic bacteria. Specificity tests were conducted with various biological molecules to evaluate the sensing performance. Results & Discussion Specific binding of LPS with peptide release the peptides from GO, resulting in fluorescence recovery, allowing ultrasensitive detection of LPS with the limit of detection of 130 pM, the most sensitive synthetic LPS sensors to-date. The LPS sensor is highly selective to LPS than other biological species. Conclusion We developed a peptide-GO assembled fluorescence sensor for ultrasensitive and specific LPS/endotoxin detection. This is the most sensitive synthetic LPS sensor reported in the world.