Fluorescent cell membrane probes (1a-d) were designed from bioactive oxazolo [4,5-b]pyridine unit. The probes were connected by oxazolo [4,5-b]pyridine and benzofuran (1a), triphenyl (1b), coumarin (1c), diethylaminobenzene (1d), respectively, with a double bond. The optical properties of 1a-d with different solvents were tested, they had obvious solvatochromism in different polar solvents, Large stokes shifts (63–204 nm) and significant fluorescent enhancement in large multilamellar vesicles (MLV). After 6 h of continuous illumination, the remaining absorption of the probes was 81–97%. The cytotoxicity test indicated that the survival rate of HeLa and L929 cells was 94–119% after co-cultured with probes 1a-d for 6 h. Laser confocal experiments further illustrated that the probes could quickly (<1 min) combine with the cell membrane. Among them, probe 1a worked in the green channel, while probes 1b, 1c and 1d employed in the red channel. Moreover, the retention time of probes 1a and 1c on cell membrane was found to 2–3 h.