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In order to monitor the intakes of plutonium by workers and estimate the internal exposure doses efficiently, a recommended technique by the international commission on radiological protection (ICRP) was measurement of activity in excreta, such as urine. The separation and purification method were provided for plutonium in urine by an automatic solid phase extraction apparatus. After the optimization of the experimental method, the whole experiment could be processed automatically. The experiments were operated in the following conditions: nitric acid concentration of 7~8 mol/L, adsorption rate of 0.5 mL/min, desorption rate of 0.3 mL/min, desorption solution of 10 mL with 0.36 mol/L HCl-0.01 mol/L HF. The whole recovery rate and relative standard deviation were 75.70% and 7.98%, respectively. It showed that the separation efficiency and the stability of samples were improved dramatically in the pre-treatment process of plutonium in urine by using automatic solid phase extraction apparatus.