Disclosed is a method of detecting a test substance contained in a sample using a working electrode and a counter electrode. The method comprises (A) forming a complex on the working electrode, the complex comprising the test substance and a metal particle; (B) washing the working electrode; (C) adding a measurement solution to the working electrode and measuring a current, voltage or electric charge from the metal on the working electrode in the measurement solution by electrochemical measurement; and (D) detecting the test substance in the sample based on a measurement result obtained in the step (C). In the method, the step (B) comprises washing the working electrode using a measurement solution having the same composition as the composition of the measurement solution used in the step (C). In the method, pH of the measurement solution is set, based on zeta potentials of the metal particle and the working electrode, to pH in which no electrostatic interaction occurs between the metal particle and the working electrode or pH in which an electrostatic repulsive force occurs between the metal particle and the working electrode.