Biological sensing technology utilizing nanoparticles extends through a diverse range of fields. The nanosensing is controlled using the assembly/disassembly of nanoparticles dominated by interaction forces between them. Although the interaction potential surface gives decisive information on the sensing mechanism, evaluating the quantitative profile has been impossible due to extremely complicated interactions of conjugated soft matter. In this study, a model-potential-free determination of the interaction potential surfaces was devised by combining small-angle scattering and liquid-state theory. The model-potential-free liquid theory was developed for colloidal nanoparticles inherently with strong van der Waals attraction forces by their nanoscopic size. The present method extracts interaction potential between nanoparticles even in systems with complicated interactions due to conjugated soft matter. By applying this determination method to a glutathione-triggered biosensing reaction, interaction potential curves between biosensing nanoparticles were realized for the first time. The analysis revealed peculiar potential surfaces of the sensing nanoparticles. The mechanism of colorimetric nanosensing function based on surface plasmon resonance is discussed from the viewpoint of the assembly/disassembly of nanoparticles in nanocomposites dominated by the interaction potential surfaces.