The method relates to a method and a corresponding kit for inhibiting nicking-mediated isothermal amplification (NEMA) DNA synthesis from the beginning. The method includes: appropriate nicking endonuclease is used in the kit, the use amount of the nicking endonuclease and dNTP is increased and auxiliary factors such as betaine and trehalose are added so as to inhibit DNA synthesis from the beginning, which causes the situation that results cannot be determined, under the premise that effective amplification is guaranteed. The kit contains reagent. By the method simple to operate, low in cost and applicable to isothermal amplification detection means, effective determination of NEMA amplification results is achieved, an inhibition effect, caused by the DNA synthesis from the beginning, on reactions is eliminated, and credibility of isothermal amplification technology is increased.