Novel methods for creating protein arrays with one- or two-dimensional control can significantly enhance basic biological research as well as various bioarray applications. We demonstrate that the structural variety and chemical heterogeneity of self-assembled, polystyrene-b-polymethylmethacrylate and polystyrene-b-polyvinylpyridine can be successfully exploited as templates for easy and rapid fabrication of functional protein arrays over a large scale. Spontaneous formation of such polymeric template-guided protein molecules yields high density protein arrays that exhibit repeat spacings in a nanoscopic dimension. The ensuing self-assembled protein molecules in the array maintain their natural conformation and activity over a very long time period. By tuning the size of the underlying block copolymer templates, our diblock copolymer-based approach to create high-density protein patterns also permits spatial control over one- or two-dimensional repeat spacings of protein nanoarrays. These unique advantages of diblock copolymer templates make the spontaneously-constructed protein nanoarrays highly suitable as functional protein sensor substrates. Therefore, our novel two-dimensional protein assembly method can be greatly beneficial for high-throughput proteomic assays and multiplexed high-density protein sensing applications.