A method for detecting a target substance in a sample, comprising steps of: mixing a first bound substance in which a first substance containing a stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance, a second bound substance in which a hydrophilic second substance binds to a second affinity substance having affinity to the target substance, and the sample; wherein the second substance is a hydrophilic polymer that is capable of inhibiting aggregation of the stimuli-responsive polymer, and the hydrophilic polymer is dextran, cyclodextrin, agarose or hydroxypropylcellulose, a polymer containing polyoxyalkylene as a constitutional unit, or polyvinyl alcohol; wherein the stimuli-responsive polymer is a temperature-responsive polymer, a pH-responsive polymer or a light-responsive polymer, and is capable of undergoing a conformation change in response to an external stimulus to aggregate; wherein the first affinity substance is a monoclonal antibody and the second affinity substance is a monoclonal antibody, and they can simultaneously bind to different sites of the target substance; placing the mixture under conditions to aggregate the stimuli-responsive polymer; wherein if the target substance is present, aggregation of the stimuli responsive polymer is inhibited by the hydrophilic moiety of the second bound substance and the stimuli-responsive polymer disperses; and if the target substance is not present, the stimuli-responsive polymer aggregates since aggregation is not inhibited; determining if the stimuli-responsive polymer is dispersed or not; and detecting the presence or absence of the target substance by observing the inhibition of aggregation. mixing a first bound substance in which a first substance containing a stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance, a second bound substance in which a hydrophilic second substance binds to a second affinity substance having affinity to the target substance, and the sample; wherein the second substance is a hydrophilic polymer that is capable of inhibiting aggregation of the stimuli-responsive polymer, and the hydrophilic polymer is dextran, cyclodextrin, agarose or hydroxypropylcellulose, a polymer containing polyoxyalkylene as a constitutional unit, or polyvinyl alcohol; wherein the stimuli-responsive polymer is a temperature-responsive polymer, a pH-responsive polymer or a light-responsive polymer, and is capable of undergoing a conformation change in response to an external stimulus to aggregate; wherein the first affinity substance is a monoclonal antibody and the second affinity substance is a monoclonal antibody, and they can simultaneously bind to different sites of the target substance; placing the mixture under conditions to aggregate the stimuli-responsive polymer; wherein if the target substance is present, aggregation of the stimuli responsive polymer is inhibited by the hydrophilic moiety of the second bound substance and the stimuli-responsive polymer disperses; and if the target substance is not present, the stimuli-responsive polymer aggregates since aggregation is not inhibited; determining if the stimuli-responsive polymer is dispersed or not; and detecting the presence or absence of the target substance by observing the inhibition of aggregation.