In this study, a phase-shifting interferometer to conduct real-time high-resolution measurements of concentration profiles in binary diffusion fields was developed. The phase-shifting interferometer comprises a Mach–Zehnder interferometer, a rotating polarizer, a CCD camera, and an image-processing unit. A phase-shifting technique was used to determine the phase difference between a test beam and a reference beam by using three images taken at intervals of 1/30 s. The phase difference is obtained for a spatial resolution of 640×240. This data is further processed in real-time to visualize the concentration profile inside a diffusion cell. The diffusion coefficient is determined by performing an inverse analysis of the experimental concentration profile. The objective function makes use of a numerical calculation, based on Fick's law, for which the initial experimental concentration profile is taken as initial condition. The diffusion field was formed inside a thermally controlled diffusion cell with optical paths as large as 20 mm. This large optical path allows measurements of diffusion fields with concentration differences as narrow as 1 mg/ml. In order to validate the measurement method, the concentration dependence of the isothermal diffusion coefficient of NaCl and Sucrose was determined in the dilute region at 25 °C, such values being extensively reported in the literature. It was found that our optical system is much faster and accurate than similar optical systems to determine the diffusion coefficients in binary systems.