The precise design of bioactive surfaces, essential for the advancement of many biomedical applications; depends on achieving control of the surface architecture as well as on the ability to attach bioreceptors to antifouling surfaces. Herein, we report a facile avenue toward hierarchically structured antifouling polymer brushes of oligo(ethylene glycol) methacrylates via surface-initiated atom transfer radical polymerization (SI-ATRP) presenting photoactive tetrazole moieties, which permitted their functionalization via nitrile imine-mediated tetrazole-ene cyclocloaddition (NITEC). A maleimide-functional ATRP initiator was photoclicked to the side chains of a brush enabling a subsequent polymerization of carboxybetaine acrylamide to generate a micropatterned graft-on-graft polymer architecture as evidenced by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Furthermore, the spatially resolved biofunctionalization of the tetrazole-presenting brushes was accessed by the photoligation of biotin-maleimide and subsequent binding of streptavidin. The functionalized brushes bearing streptavidin were able to resist the fouling from blood plasma (90% reduction with respect to bare gold). Moreover, they were employed to demonstrate a model biosensor by immobilization of a biotinylated antibody and subsequent capture of an antigen as monitored in real time by surface plasmon resonance.