In this work, we present theoretical and experimental studies of nanofluidic channels as a potential biosensor for measuring rapid protein complex formation. Using the specific properties offered by nanofluidics, such as the decrease of effective diffusion of biomolecules in confined spaces, we are able to monitor the binding affinity of two proteins. We propose a theoretical model describing the concentration profile of proteins in a nanoslit and show that a complex composed by two bound biomolecules induces a wider diffusion profile than a single protein when driven through a nanochannel. To validate this model experimentally, we measured the increase of the fluorescent diffusion profile when specific biotinylated dextran was added to fluorescent streptavidin. We report here a direct and relatively simple technique to measure the affinity between proteins.