Integrated-optical difference interferometers – operated at a single wavelength – were developed in our group as biosensors for monitoring bio-specific interactions in real time. Their potentially very high resolution cannot be exploited in practice, because refractive-index and/or temperature variations of the sample solutions also contribute to the sensors' response, i.e., to the time-dependent phase difference Δ Φ ̃ (t) between the TE 0 and TM 0 modes excited in a planar or rib waveguide after their polarization-dependent interaction with the sample. In single-wavelength operation of the difference-interferometric biosensor we show experimentally and theoretically that its cross-sensitivities for refractometric perturbations can be greatly reduced by optimisation of the waveguide thickness. We also investigate how the temperature sensitivity of the biosensors depends on the waveguide thickness. We demonstrate that a difference interferometer, where the phase shifts Δ Φ ̃ (t) are determined from the lateral displacements of spatial interference fringes, can be simultaneously operated – without any additional hardware components – at two laser wavelengths λ 1 and λ 2; in our experiments with 80–160 nm thick Si 3N 4 planar or rib waveguides we used the wavelengths λ 1=633 nm and either λ 2=491 or 488 nm, respectively. We show that from the two independent sensor responses, viz., the two phase shifts Δ Φ ̃ λ 1 (t) and Δ Φ ̃ λ 2 (t) , the biosensing effect, can be differentiated from simultaneously occurring time-dependent refractive-index and/or temperature variations of the sample. As an experimental test of dual-wavelength operation in biosensing, we monitor the affinity binding between avidin and biotinylated proteins, with superimposed refractive-index and/or temperature variations as perturbations.