We present a novel label-free electrokinetic method for detecting biomolecular binding on nanoparticles suspended in the vicinity of a laminar microfluidic interface. The sensor is based on the deflection of the liquid interface in an external AC electric field. Biomolecular binding on particles is shown to increase the interfacial electrical conductivity of the suspending electrolyte, which is sensitively transduced as a change in electrokinetic interfacial deflection. Using this approach, we detect the binding of biotin on streptavidin-functionalized nanoparticles at concentrations as low as 500 aM and perform detection of human IgG at clinically relevant concentrations (1.25–12 mg/mL) without labels. The interface response is only influenced by specific binding on nanoparticle binding sites and decreases when the particle concentration of reactive particles is reduced. Furthermore, we show that control experiments with both non-reactive particles and lack of a specific target analyte do not produce interfacial deflection. This work provides a promising method for quantifying bead-based binding kinetics for sensitive and specific biosensing in solution without labels.