Biosensing technology represents a superior way of identifying a wide range of biomolecules that exist in the human body, food, water, and other environmental sources. In this regard, human health issues are a growing global concern, and the development of effective methods for the detection and differentiation of biomolecules during diagnosis and/or treatment of diseases is essential. However, achieving adequate levels of sensitivity and specificity will be fundamental for the successful clinical application of biosensing technology. Notably, sensitivity and specificity are highly dependent on non-fouling (specificity) of biomolecules on the sensing surfaces, particularly with regard to nanoparticle-tagged biomolecules. Here, the authors have evaluated bio-fouling (i.e., biological non-specificity) by analysing four distinct sensing substrates (gold, silicon, silica and platinum) using anti-mouse immunoglobulin-conjugated gold nanoparticles. As evidenced by scanning electron microscopy and ultraviolet-visible-near-infrared recording spectrophotometry, silica (SiO 2 ) lacked bio-fouling, whereas gold (Au) showed high bio-fouling potential. Indeed, Raman spectroscopic analysis further confirmed these results. Thus, the present findings indicate that SiO 2 is an optimal substrate for biosensor development.