The aggregation-induced emission (AIE) of a 1,2-diphenyl-1,2-di(p-tolyl)ethene (TPE) was explored as a novel fluorescence method for probing the assembling/disassembling of amphiphilic molecules. The fluorescence intensity was able to monitor the formation of micelles and determine the critical micelle concentration (CMC) of surfactants. The temperature-dependent micellization of the pharmaceutically important PEO–PPO–PEO copolymer, Pluronic F127, was further studied by using the TPE fluorescence spectrum intensity. Our results showed good agreement with those reported in the literature by using other methods. The special advantage of the AIE probe method was further explored to determine the assembling/disassembling process of the colored amphiphilic molecule, 1-[4-(3-phenylazophenoxy)butyl]triethylamine bromide (AzoC4), whose CMC value has not previously been described. Since the TPE fluorescence signal mainly comes from the aqueous phase, not from the inside of hydrophobic core, it provides a possible platform to study the CMC of those colored surfactants. Based on the novel fluorescence properties of TPE in the aggregated and dispersed states, one can conclude that the TPE method is a promising method for the determination of the CMC and critical micellization temperature (CMT), particularly having a special advantage to determine the assembling/disassembling process of colored amphiphilic molecules.