A new technique is developed to measure the nanoparticles' Brownian motions by employing microparticle-tracking velocimetry (micro-PTV) in evanescent wave field, which can provide high signal-to-noise ratio images for analyzing nanoparticles. movements. This method enables real-time detection of C-reactive proteins (CRPs) during the rapid interaction between CRPs and anti-CRP-coated nanobeads as CRP concentrations are related to the nanobeads' Brownian velocity in the equilibrium state. The smallest observable nanobeads with 185 nm were utilized in this experiment to detect CRP concentrations as low as 0.1 mu g/mL. even in a high-viscosity solution. Further, the dissociation constant, K-D, can be evaluated based on the experimental results.