Abstract: Objective: An intravital fluorescence microscopic method and digital image analysis were established for measuring the changes of fluorescein sodium （FlNa, MW 376） permeability in mesenterial microvessels. Methods: The transport of fluorescent tracer within the blood vessels and through the vessel walls into the perivascular tissue of the rat mesentery was study using a highly sensitive CCD camera, the fluorescence intensities were transformed into video signals and recorded for subsequent analysis. Results: The results show that FlNa can pass through the wall of microvessels with minimal delay. The permeabiity velocity was a constant （1.1635×10 ^-7 cm ²/s） and very fast within the first 20 secend, after that,the velocity was 5 times slower and still a constant （0.2045×10 ^-7 cm ²/s）. Conclusion: it is expected that this method would be very useful for the quantitive analysis in various molecular weights and in monolayer endothelial cells permeability.