A novel fluorescence-based array biosensor targeted for field applications, such as environmental monitoring, has been developed, and successfully applied to DNA hybridization assays. The purpose was to meet the demand for automated, portable but easy-to-maintain systems allowing continuous flow monitoring of surface reactions. The biosensor presented here can be distinguished from the existing systems by the optical method used, which provides an enhanced simplicity and robustness, and enables a simple maintenance by potentially unskilled personnel. The system is based on a conventional microscope slide which acts both as transducer and biological array sensor. The excited fluorescence is guided by total internal reflection into the slide to the detector which is directly interfaced to the slide. Each region of the sensor array is successively optically interrogated, and the detection of the corresponding fluorescent emission synchronized. A real-time three-analyte analysis is thus feasible without any mechanical scanning movement or optical imaging systems as generally used in the existing instruments. The ability of the biosensor to operate in continuous flow for several tens of hours has been demonstrated. The biosensor has been assessed in terms of stability, and slide-to-slide reproducibility, which is found to be less than 3.7%, thus far below the standard biological reproducibility. DNA hybridization assays were performed to estimate a limit of detection, which was found to be 16 mol/microm(2), and to determine the reaction kinetics associated to the DNA model used. The developed biosensor is thus shown to be able to predict reaction kinetics, and to monitor in real time surface reactions between targets and probes.