In this work, 5-Fu-loaded PLLA microparticles were prepared by a two-step method combining supercritical antisolvent (SAS) process and supercritical solvent impregnation (SSI) process. The PLLA microparticles with mean particle size of 0.68 mu m were prepared by SAS process, then the drug of 5-Fu were impregnated in PLLA microparticles by SSI process. The effects of cosolvent concentration, pressure, and temperature on the morphology of microparticles and the drug impregnation amount were investigated. The morphology of microparticles was studied by scanning electron microscope (SEM), and the impregnation amount of drug was evaluated by drug release method. The results showed that the morphology of drug-loaded polymer microparticles was determined by SAS process and had nothing to do with SSI process in the two-step method. The impregnation amount of drug was higher in the case with cosolvent than that without cosolvent under the same conditions. With the increase of the cosolvent concentration, the impregnation amount of drug in the PLLA microparticles increased first and then decreased, while it increased with the decrease of pressure and the increase of temperature. By the two-step method with cosolvent, 5-Fu-loaded PLLA microparticles could be successfully prepared and the impregnation amount of drug could increase effectively.