The competitive adsorption of methylene blue (MB) and beta-casein on hydrophilic glass from an aqueous mixed solution was directly detected at the solution pH smaller than the protein isoelectric point (pI) by means of the waveguide-based broadband time-resolved evanescent wave absorption spectroscopy. The competitive adsorption causes the MB coverage to exponentially decrease with time from its peak value and prevents MB aggregation at the interface. The kinetic equation for the competitive adsorption of binary adsorbates was theoretically deduced based on the Langmuir model, and was used for creating the best fit to the experimental data. In the case of a fixed concentration of MB in the mixed solution, the best-fit parameter tau(-1) increases with the protein concentration at a specific pH and decreases with the solution pH at a given concentration of protein. The findings suggest that the beta-casein concentration in sub-mu M level can be rapidly determined by the time-resolved waveguide absorptiometry based on the competitive adsorption of MB and protein.