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We present a simple and an inexpensive method for the fabrication of a nano-scale fluidic filter in a local region of a microchannel by self-assembly of nano-spheres and surface tension. In this work, we use colloid-plug that is formed by surface tension of liquid in a microchannel. When colloid is evaporated, nano-spheres in a colloid are orderly stacked by a capillary force. Orderly stacked nano-spheres form 3-D nano-mesh which can use as a mesh structure of a fluidic filter. We used silica nano-sphere whose diameter is 567±185nm, and silicon microchannel of 50 μm-diameter. Fabricated nano-fluidic filter in a microchannel has median pore diameter of 157nm which was agreement with 113±37nm of 20% of the sphere diameter. From experimental study, we verified a new fabrication method of a nano-fluidic filter in a local region of a microchannel.