Novel strategies of developing fluorescent sensors for proteins are highly demanded. In this work, we particularly synthesized a cholesterol-derivatized pyrene probe. Its fluorescence emission is effectively tuned by the aggregation state of a cationic surfactant dodecyltrimethylammonium bromide (DTAB). The used probe/DTAB assemblies exhibit highly sensitive ratiometric responses to pepsin and ovalbumin egg (o-egg) with detection limits of 4.8 and 18.9 nM, respectively. The fluorescence changes indicate the protein-surfactant interaction leads to further aggregation of DTAB assemblies. The results from Tyndall effect and dynamic light scattering verify this assumption. The responses to pepsin and o-egg are due to their strong electrostatic or hydrophobic interaction with DTAB assemblies at pH 7.4. The present noncovalent supramolecular sensor represents a novel and simple strategy for sensing proteins, which is based on the encapsulated fluorophore probing the aggregation variation of the surfactant assemblies.