In this paper, the implementation and characterization of a hand-held and simple biosensor for in-situ endotoxin determination are described. The integrated biosensor developed here is based on the electrochemical detection of endotoxin using polymyxin B as bioreceptor immobilized onto gold electrodes via a self-assembled monolayer. The cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy were used to characterize the biosensor performance and properties throughout the functionalization process. In addition, a comparative analysis of the behavior and features of two alternative electrochemical techniques for endotoxin detection was carried out. The biosensing device fabricated by thin-film technologies provided a simple and robust method to detect low concentrations of endotoxin.