Since most clinical assays are performed on cell-free serum or plasma, micro-analytical systems for blood tests require integrated on-chip microfluidics for the isolation of plasma or serum from crude blood samples. In this paper, we present a crossflow filtration method using novel silicon nano-filters for plasma separation. The microfluidic chip is made of a silicon substrate containing micropillar arrays, feed channels, side channels and nano-gap structures, sealed with a PDMS-glass compound cover. The design of the silicon filtration structures were optimized using numerical analysis and the optimal MEMS fabrication procedures were obtained. The filtration structures including nano-filters were characterized using SEM and subsequently used to isolate plasma from whole blood in a continuous manner. Compared with micro-gap structures in silicon microfluidic channels, the nano-gap structures have been used to separate plasma from whole blood samples with higher selectivity, where a maximum plasma selectivity of 97.7% has been obtained. Common problems of clogging and jamming in filtration applications have seldom been noticed in our device. The presented microfluidic filtration device for plasma isolation could be integrated into JAS for point-of-care diagnostics in the near future.