PURPOSE: An electrochemical one-step nucleic acid detection and quantitation method by controlling the transport of signaling materials is provided to simultaneously detect various kinds of target nucleic acids using an electrochemical one-step nucleic acid detection kit. CONSTITUTION: An electrochemical one-step nucleic acid detection and quantitation method by controlling the transport of signaling materials comprises the steps of: adding a target nucleic acid to a one-step nucleic acid detection kit and hybridizing the target nucleic, a capture probe, and a signaling probe; applying electric current to the kit and measuring a change of the electric current to detect and quantitate the target nucleic acid. The capture probe is fixed on the bottom surface of a reaction chamber and has a sequence which is complementary to a target nucleic acid sequence. The signaling probe is labeled with a signal substance which generates an electrochemical signal by reduction and oxidation and has a sequence which is complementary to another target nucleic acid sequence. The kit contains an analysis solution in which the signaling probe is dissolved and has a chamber with a working electrode, a reference electrode, and an auxiliary electrode. [Reference numerals] (AA) Metal nanoparticle-labeled signaling probe; (BB) Glass carbon electrode; (CC) Signal; (DD) Target nucleic acid; (EE) Gold matrix; (FF) Analysis chip