Herein, we report a bottom-up approach to assemble a series of stochastic DNA walkers capable of probing dynamic interactions occurring at the bio–nano interface. We systematically investigated the impact of varying interfacial factors, including intramolecular interactions, orientation, cooperativity, steric effect, multivalence, and binding hindrance on enzymatic behaviors at the interfaces of spherical nucleic acids. Our mechanistic study has revealed critical roles of various interfacial factors that significantly alter molecular binding and enzymatic behaviors from bulk solutions. The improved understanding of the bio–nano interface may facilitate better design and operation of nanoparticle-based biosensors and/or functional devices. We successfully demonstrate how improved understanding of the bio–nano interface help rationalize the design of amplifiable biosensors for nucleic acids and antibodies.