A nanopore sensor comprises second electrophoresis electrode or micropump, second fluidic reservoir, second micro-nanometer separation channel, substrate, sub-nanometer-thick functional layer, first micro-nanometer separation channel, first electrophoresis electrode or micropump, and first electrophoresis electrode or micropump that are sequentially assembled. An opening and a nanopore are provided through the substrate and the sub-nanometer-thick functional layer, respectively. A first electrode for measuring ionic current is provided in the first micro-nanometer separation channel, and a second electrode for measuring ionic current is provide in the second micro-nanometer separation channel. The present invention provides a simple method to prepare a sub-nanometer functional layer having a nanopore extending through the sub-nanometer-thick functional layer. The pore size is comparable to the spacing between two adjacent bases in a DNA strand required for single-base resolution sequencing. The shape of nanopore overcomes nucleotide conformation effect on the identification as bases translocate through the nanopore.