Bio-nanopatterning of surfaces is a very active interdisciplinary field of research at the interface between biotechnology and nanotechnology. Precise patterning of biomolecules on surfaces with nanometre resolution has great potential in many medical and biological applications ranging from molecular diagnostics to advanced platforms for fundamental studies of molecular and cell biology. Bio-nanopatterning technology has advanced at a rapid pace in the last few years with a variety of patterning methodologies being developed for immobilising biomolecules such as DNA, peptides, proteins and viruses at the nanoscale on a broad range of substrates. In this review, the status of research and development are described, with particular focus on the recent advances on the use of nanolithographic techniques as tools for biomolecule immobilisation at the nanoscale. Present strengths and weaknesses, as well future challenges on the different nanolithographic bio-nanopatterning approaches are discussed.