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We report here a microfabricated nanofilter array chip that can size-fractionate sodium dodecyl sulfate (SDS)-protein complexes and small DNA molecules based on the Ogston sieving mechanism. Nanofilter arrays with a gap size of 40–180 nm were fabricated and characterized. Complete separation of SDS-protein complexes and small DNA molecules were achieved in several minutes with a separation length of 5 mm. The fabrication strategy for the nanofilter array chip allows further increasing of the nanofilter density and decreasing of the nanofilter gap size, leading, in principle, to even faster separation.