Abstract: A method of manufacturing a three-dimensional nanochannel device is provided. In the method, a first insulation layer is formed on a substrate, a first opening is formed in the first insulation layer, and a patterned photoresist is formed on the first insulation layer. The patterned photoresist includes at least one second opening, wherein the second opening is adjacent to the first opening and exposes the first insulation layer. Afterwards, the first insulation layer is etched and the substrate is also continued to be etched by using the patterned photoresist as a mask, so as to form a housing space, wherein a depth of the housing space is at least two orders greater than a thickness of the first insulation layer. Thereafter, the patterned photoresist is removed, and a second insulation layer is formed on a surface of the substrate.