Innovative modes of response can greatly push forward chemical sensing processes and subsequently improve sensing performance. Classical chemical sensing modes seldom involve the transition of a delicate molecular assembly during the response. Here, we display a sensing mode for polyamine detection based on an order–order transition of iron–sulfur complexes upon their assembly. Strong validation proves that the unique order–order transition of the assemblies is the driving force of the response, in which the polyamine captures the metal ion of the iron–sulfur complex, leading it to decompose into a metal–polyamine product, accompanied by an order–order transition of the assemblies. This mechanism makes the detection process more intuitive and selective, and remarkably improves the detection efficiency, achieving excellent polyamines specificity, second-level response, convenient visual detection, and good recyclability of the sensing system. Furthermore, this paper also provides opportunities for the further application of the iron–sulfur platform in environment-related fields.