Patterning nanoscale protein gradients is crucial for studying a variety of cellular processes in vitro. Despite the recent development in nano-fabrication technology, combining nanometric resolution and fine control of protein concentrations is still an open challenge. Here, we demonstrate the use of thermochemical scanning probe lithography (tc-SPL) for defining micro- and nano-sized patterns with precisely controlled protein concentration. First, tc-SPL is performed by scanning a heatable atomic force microscopy tip on a polymeric substrate, for locally exposing reactive amino groups on the surface, then the substrate is functionalized with streptavidin and laminin proteins. We show, by fluorescence microscopy on the patterned gradients, that it is possible to precisely tune the concentration of the immobilized proteins by varying the patterning parameters during tc-SPL. This paves the way to the use of tc-SPL for defining protein gradients at the nanoscale, to be used as chemical cues e.g. for studying and regulating cellular processes in vitro.