In order to characterize the photon luminescence properties of up-conversion nanoparticles(UCNPs), a system to measure fluorescence life time of single nanoparticle was established based on a custom-built two-photon microscope scanned with a pair of galvo-mirrors. First, a sample of mono-disperse UCNPs was imaged by the two-photon microscope. Then, the exact location of each nanoparticle was precisely determined through the single molecule fluorescence localization algorithm. The laser was focused on the nanoparticles one by one with a laser pulse with 500 μs duration exerted on the nanoparticle. A photomultiplier was employed to measure the time-lapse fluorescence intensity which varied with time. Finally, the fluorescence decay curve was fitted to calculate the life time of single nanoparticle. The experiment results show that the fluorescence curves of single nanoparticle fit with single exponential decay, and the fluorescence life time is 195.3 μs. For contrast, the life time of the bulk sample is 358.9 μs. This indicates a dramatic effect of particle aggregation on the photon luminescence properties of UCNPs.