Abstract: In order to determine the concentration of uranium and plutonium in 1AP of bench experiment in time, an on-line X-ray fluorescence spectrometer was designed and set up, and the angle between incident X-ray and emitted X-ray was 60°. The spectrometer was airproofed in order to protect the X-ray tube and the detector. Based on the plotted working curve of uranium and plutonium, the analytical method was established to determine the concentration of uranium and plutonium in 1AP. The relative standard deviation of the measured concentration of uranium is 0.42%. The relative range for repeated measurement of uranium concentration in three hours is 1.6%. The results show that the device is stable enough. This spectrometer was applied in on-line determination of uranium. It can monitor the fluctuation of uranium concentration, and the results agree well with that of titration means.