Provided are a method of detecting a target substance, by which the detection sensitivity in the PALSAR method can be improved and multiple genes can be simultaneously detected, and a kit for detection. The method is a method of detecting a target substance by forming a signal probe polymer with the use of multiple dimer probes or dimer-forming probes and an assist probe, in which each dimer probe is made up of two kinds of dimer-forming probes including a 5′-side region, a central region and a 3′-side region (the central regions of the two kinds of dimer-forming probes are complementary to each other and the 3′-side regions and the 5′-side regions thereof are not complementary to each other), the multiple dimer probes are constituted such that a dimer probe polymer is formed via a self-assembly reaction, and the assist probe is designed so as to have a structure having a region complementary to the 5′-side region of one dimer-forming probe in one dimer probe, a region complementary to the 3′-side region of the other dimer-forming probe, and a target region capable of binding to the target substance.