Polydiacetylene (PDA) has been widely used as a biosensor candidate because it exhibits specific blue-red transition upon varied external stimulations, such as heat, pH, or mechanical pressure. We prepared mixed vesicles using the 10,12-pentacosadiynoic acid (PCDA) and fluorescence probe BO558, and investigated the interaction of surfactant with PCDA/BO558 vesicles. Experiments exhibited that the fluorescence of BO558 was significantly quenched owing to the ene-yne conjugated backbones in the PCDA vesicles. However, the fluorescence was gradually recovered in the presence of cetyltrimethylammonium bromide (CTAB) to the system. Meanwhile, the turbidity of the mixed solution decreased greatly with the increase of CTAB concentration. We investigated the mechanism of the fluorescence quenching and recovery in the system of PCDA/BO558 vesicles and CTAB by UV-vis spectrum, stable fluorescence, transmission electron microscopy. The morphology of vesicles transformed with the change of surfactant concentration.