The present invention provides a method for analyzing a template nucleic acid, a method for analyzing a target substance, an analysis kit for a template nucleic acid or a target substance, and an analyzer for a template nucleic acid or a target substance, which are excellent in accuracy. The method for analyzing a template nucleic acid of the present invention includes the steps of: fractionating a sample containing a template nucleic acid into a plurality of template nucleic acid fractions; amplifying a target sequence and its complementary sequence in the template nucleic acid with respect to each of the plurality of template nucleic acid fractions in the presence of a nucleic acid amplification reagent; detecting generation or quenching of a signal that shows an amplification of the target sequence or the complementary sequence with respect to each of the plurality of template nucleic acid fractions after the amplification step; and discriminating a template nucleic acid fraction in which the generation or quenching of a signal that shows the amplification has been detected among the plurality of template nucleic acid fractions as an amplified fraction in which the target sequence or the complementary sequence has been amplified, wherein the nucleic acid amplification reagent contains a primer set that amplifies the target sequence and the complementary sequence and a signal generating substance that generates or quenches a signal in response to the amplification, and the signal generating substance generates a signal in a state where it is bound sequence-dependently and quenches a signal in a state where it is not bound or quenches a signal in a state where it is bound sequence-dependently and generates a signal in a state where it is not bound, and generation and quenching of a signal are reversible.