The present invention relates to a method of screening a target metabolite-overproducing mutant strain using a synthetic suicide genetic circuit, and more particularly to a method of screening only a metabolite-overproducing mutant strain while killing a mutant strain that does not produce the metabolite, by introducing into mutant strains a synthetic suicide genetic circuit comprising a suicide gene coupled with a riboswitch. The method for screening a target metabolite-overproducing mutant strain according to the present invention has advantages in that a metabolite-overproducing mutant strain having a relatively fast or slow growth rate can be separated by visual observation, and in that the riboswitch that is used in the synthetic suicide genetic circuit can be replaced depending on the kind of target metabolite, and thus the synthetic suicide genetic circuit can be applied commonly to various strains.