In this paper an electrochemical endotoxin biosensor consisting of an immobilized lipopolysaccharide (LPS) ligand, polymyxin B (PmB), is presented. Several parameters involved both in the device fabrication and in the detection process were analyzed to optimize the ligand immobilization and the interaction between PmB and LPS, aiming at increasing the sensitivity of the sensor. Di?erent electrochemical pre-treatment procedures as well as the functionalization methods were studied and evaluated. The use of a SAM (self-assembled monolayer) to immobilize PmB and the quanti?cation of the interactions via cyclic voltammetry allowed the development of a robust and simple device for in situ detection of LPS. Thus, the biosensor proposed in this work intends an approach to the demanding needs of the market for an integrated, portable and simple instrument for endotoxin detection.