A new on-chip whole blood/plasma separator driven by asymmetric capillary forces, which are produced through a microchannel with sprayed nanobead multilayers, has been designed, fabricated and fully characterized. The silica nanobead multilayers revealing as superhydrophilic surfaces have been fabricated using a spray layer-by-layer (LbL) nano-assembly method. This new on-chip blood plasma separator has been targeted for a sample-to-answer (S-to-A) microfluidic lab-on-a-chip (LOC) toward point-of-care clinical testing (POCT). Effective plasma separation from undiluted whole blood was achieved through the microchannel which was composed of asymmetric superhydrophilic surfaces with a 10 mm hydrophobic patch. Blood cells were continuously accumulated over the hydrophobic patch while the blood plasma was able to flow over the patch. Therefore, the blood plasma was successfully separated from the whole blood throughout the accumulated blood cells which worked as a so-called 'self-built-in blood cell microfilter'. The separated plasma was approximately 102 nL from a single drop of 3 μL whole blood within 10 min, which is very suitable for single-use disposable POCT devices.