The present invention relates to a nanopore manufacturing method, a nanopore manufactured by the manufacturing method, a real time molecular sequence analysis system using the nanopore, and a method of analyzing molecular sequences of the biopolymer using the nanopore. More particularly, the present invention relates to a method of manufacturing a nanopore, comprising: forming a first thin film on a top surface of a substrate; Removing the first thin film and a portion of the substrate using a lithography process and an etching process to form an alignment mark on the outer periphery of each of the upper and lower surfaces of the substrate on which the first thin film is formed; Etching the center side of the lower surface of the substrate until the center of the lower surface of the first thin film is exposed using a lithography process and an etching process; Forming a nano hole at the center of the first thin film using a lithography process and an etching process; Depositing a second thin film one or more times to reduce the diameter of the nano holes; Placing a third thin film layer on top; Forming a fourth thin film on the upper surface of the third thin film to protect the third thin film; Forming an nano-pore at the center of the third thin film by applying an etching process from the bottom of the substrate; And partially etching the second thin film and the fourth thin film.
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