The cell membranal liquid-ordered (Lo) phase can control the structure and function of cell membranes. In this study, we have engineered a novel two-photon (TP) fluorescent probe, TP-HVC18, which remarkably displayed two different fluorescence emission profiles in the aggregate and solution states in distinct polar environments. In accordance with its aggregate fluorescence, TP-HVC18 also can emit a red fluorescence signal in Lo phase vesicles. Taking advantage of this unique feature, we have demonstrated that the new TP probe TP-HVC18 is suitable for imaging membranal Lo phase by an aggregate fluorescence method. Furthermore, the robust probe also exhibited uncontinuous red fluorescence distribution in the cell membranal Lo phase. Based on this intriguing character, we also successfully showed that the novel probe can be employed to exhibit uncontinuous distribution of cell membranal Lo phase by a 3D imaging technique. We expect that this aggregation-based fluorescent platform may be extended for the development of a wide variety of TP fluorescent probes for detecting several biological species.