It is my honor to introduce our recent research on pushing the boundaries of antifouling bio-functional materials and nano-coatings, with a particular focus on enhancing biosensing in real-world media. To address this challenge, we explore the fascinating properties of ultrathin antifouling bio-functional polymer brushes. Namely, we will share recent advances in the following areas: i) design, fabrication, and responsive behavior of antifouling functionalizable brush nano-coatings, ii) molecular-level research on biofunctionalization, iii) the integration of zwitterionic brushes in advanced cell-on-a-chip systems, and iv) examples of antifouling point-of-care biosensors with tailor-made microfluidics systems. Results show how important (and challenging) it is to precisely control over the antifouling properties of polymer nano-coatings when submerged in real-world complex biological fluids. We detail new approaches to probe brushes with emerging electrochemical impedance spectroscopy technique. We present a new antifouling terpolymer brush with a superior combination of antifouling and bio-functionalization capabilities over similar bio-functional coatings. This material has been successfully employed in several antifouling biosensors; for example, in piezoelectric biosensors for the rapid sensitive detection of SARS-CoV-2 in complex clinical and surface swab samples as well as bacterial pathogens in fresh foods.