Diseases and epidemics caused by pathogens in drinking water supplies not only cause deaths but also lead to an important economic and labor loss. Rapid and easy detection of these pathogens on water sources will help us to prevent the diseases associated with them. In this study, the aim is to achieve the detection of low amount of toxins by amplification of the signal by surface plasmon fluorescence spectroscopy (SPFS) and to minimize the need for sample preparation. Cholera toxin B (ChB), a truncated form of cholera toxin, was chosen as the model toxin. Capture antibody that will assure specific binding of the toxin was immobilized to the sensor surface by different methods and measurement conditions were optimized.