Novel methods for affixing functional proteins on surfaces with high areal density have the potential to promote basic biological research as well as various bioarray applications. The use of polymeric templates under carefully balanced thermodynamic conditions enables spontaneous, self-assembled protein immobilization on surfaces with spatial control on the nanometer scale. To assess the full potential of such nanometer-scale protein platforms in biosensing applications, we report for the first time the biological activity of proteins on diblock copolymer platforms. We utilized horseradish peroxidase, mushroom tyrosinase, enhanced green fluorescent protein, bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine IgG, and protein G as model systems in our protein activity studies. When specific catalytic functions of HRP and MT, immobilized on selective domains of microphase-separated PS-b-PMMA, are evaluated over a long period of time, these enzymes retain their catalytic activity and stability for well over 3 months. By performing confocal fluorescence measurements of self-fluorescing proteins and interacting protein/protein systems, we have also demonstrated that the binding behavior of these proteins is unaffected by surface immobilization onto PS-b-PMMA diblock copolymer microdomains. Our polymer platforms provide highly periodic, high-density, functional, stable surface-bound proteins with spatial control on the nanometer scale. Therefore, our diblock copolymer-guided protein assembly method can be extremely beneficial for high-throughput proteomic applications.