Immunoassay has become a versatile tool in several fields of analytical chemistry. We describe the characterization and the application of different integrated optical channel waveguide Mach-Zehnder interferometers (MZIs) as label-free immunoprobes. The performance of the classical MZI is compared with that of a modified structure which incorporates a 3 × 3 coupler. Characterization of the devices demonstrates a dramatic improvement gained by using the 3 × 3 coupler. Two main advantages are achieved by the modified device. First, the possibility of referencing the output signal allows the elimination of signal fluctuations due to coupling and light-source instabilities. An increase of the signal-to-noise ratio by a factor of up to 10 is achieved. Secondly, the phase shift between the three outputs allows unambiguous detection with optimum sensitivity. For the detection of the herbicide simazine, the functional properties of the transducer surface are optimized by an appropriate chemical modification. Using this improved device, a simazine immunoassay has been carried out with a test midpoint of 0.3 ppb and a detection limit of approximately 0.1 ppb. The excellent performance, established manufacturing techniques and the potential for simplification and parallelization make the device attractive for further development.