A biosensor system based on total internal reflectance fluorescence (TIRF) was used to discriminate a mixture of the triazines atrazine and simazine. Only cross-reactive antibodies were available for these two analytes. The biosensor is fully automated and can be regenerated allowing several hundreds of measurements without any user input. Even a remote control for online monitoring in the field is possible. The multivariate calibration of the sensor signal was performed using artificial neural networks, as the relationship between the sensor signals and the concentration of the analytes is highly non-linear. For the development of a multi-analyte immunoassay consisting of two polyclonal antibodies with cross-reactivity to atrazine and simazine and different derivatives immobilised on the transducer surface, the binding characteristics between these substances like binding capacity and cross-reactivity were characterised. The examination of three different measurement procedures showed that a two-step measurement using only one antibody per step allows a quantification of both analytes in a mixture with limits of detection of 0.2 microg/l for atrazine and 0.3 microg/l for simazine. The biosensor is suitable for online monitoring in the field and remote control is possible.