Exposure to pathogens in recreational or drinking water is a serious public health concern. It is important to rapidly determine and identify trace levels of pathogens in real environmental samples. We report here on a label-free potentiometric aptasensor for rapid, sensitive, and selective detection of Listeria monocytogenes (LM), a pathogen widely distributed in the environment. An aptamer binds specifically to internalin A, a surface protein present in LM cells. The target-binding event prevents the aptamer from electrostatically interacting with protamine, which can be sensitively detected using a polycation-sensitive membrane electrode. Using this method, LM can be detected down to 10 CFU mL(-1). Coupled to an online filtration system, the bioassay has been evaluated with spiked coastal seawater samples and shows good recovery and high accuracy. This work demonstrates the possibility of developing potentiometric aptasensors for determination and identification of various bacteria in environmental samples.