A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell. Dual chemical functionalization of graphene oxide (GO) with Tris- [nitrilotris(acetic acid)] and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is described. The resulting nanoparticles are non-cytotoxic and can simultaneously deliver oligohistidine- and biotin-tagged biomolecules into the cell. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.