Class I hydrophobin Vmh2, a peculiar surface active and versatile fungal protein, is known to self-assemble into chemically stable amphiphilic films, to be able to change wettability of surfaces, and to strongly adsorb other proteins. Herein, a fast, highly homogeneous and efficient glass functionalization by spontaneous self-assembling of Vmh2 at liquid–solid interfaces is achieved (in 2 min). The Vmh2-coated glass slides are proven to immobilize not only proteins but also nanomaterials such as graphene oxide (GO) and quantum dots (QDs). As models, bovine serum albumin labeled with Alexa 555 fluorophore, anti-immunoglobulin G antibodies, and cadmium telluride QDs are patterned in a microarray fashion in order to demonstrate functionality, reproducibility, and versatility of the proposed substrate. Additionally, a GO layer is effectively and homogeneously self-assembled onto the studied functionalized surface. This approach offers a quick and simple alternative to immobilize nanomaterials and proteins, which is appealing for new bioanalytical and nanobioenabled applications.