Here, we develop a new nanofilter device for the rapid and efficient separation of nanoparticles and biomolecules, exploiting the use of AAO mebrane with ordered nanopores in the range from 20 nm to 200 nm. Briefly, the chip comprises of a series of the upper and lower PDMS channels containing embedded inlet and outlet ports, and 50 μm width microfluidic channel, and AAO membrane to be made the filtering zone. After assembling these components, the acrylate plastic plates were used to fix the device on the top and bottom side. When introducing the samples into the inlet ports of the upper PDMS channel, we were able to separate and concentrate the nanoparticles and target molecules at the filtering zone, and to elute the solutions containing the unwanted materials toward the lower PDMS channels normal to the direction of AAO membrane. To demonstrate the usefulness of the device we apply it to the SERS detection of nucleic acid sequences associated with Dengue virus serotype 2. We report a limit of detection for Dengue sequences of 300 nM and show excellent enhancement of Raman signals from the filter zone of the nanofilter device.