Anti-fouling surfaces have been widely studied for their importance in medical devices and industry. Anti-fouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm2 which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e. an ultimate resolution) to study anti-fouling properties of functionalized surfaces. We found that about 600 Immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ~ 5 pg/cm2 adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new anti-fouling platform that exhibits improved anti-fouling performance that shows only 78 IgG molecules adsorbed (~ 0.5 pg/cm2). The anti-fouling platform consists of forming one-nm TiO2 thin layer, on which peptidomimetic anti-fouling polymer (PMAP) is robustly anchored. The unprecedented anti-fouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others.