Self-assembly of two-dimensional graphene nanosheets is an important strategy for obtaining graphene architectures for practical applications. However, construction of graphene self-assembled architecture in the presence of protein and copper salts has never been realized. In this paper, a one-pot facile, self-assembled graphene oxide (GO) with tremella-like structure in the presence of lysozyme and copper phosphate was reported. Compared with individual free lysozyme, GO, and even copper phosphate, GO/lysozyme nanocomposites showed excellent antibacterial activity against Escherichia coli, which is about ten-fold higher than that of the free enzyme. This study could provide a new method of three-dimensional self-assembled graphene oxide in the presence of protein and copper phosphate.