Interferon-alpha2b (IFN alpha-2b) microspheres were prepared at various concentrations (5%, 10%, 15%, 20% and 25%) and viscosities (0.39, 0.6, 0.89 and 1.13 dL/g) of poly(lactic-co-glycolic acid) (PLGA) using double emulsion solvent evaporation. The optimal formulation of IFN alpha-2b microspheres was determined to be 0.89 dL/g PLGA, as assessed by the in vitro release test. The pharmacokinetics of IFN alpha-2b microspheres was investigated. Nine groups of rats were injected intramuscularly with three doses (0.5, 1 and 2 MIU) of commercial lyophilized IFN alpha-2b injection or IFN alpha-2b microspheres. At a dose of 0.5 MIU, the IFN alpha-2b microsphere released significantly longer than that of the IFN alpha-2b injection. At a dose of 2 MIU, each pharmacokinetics parameter of microspheres prepared with the IFNa-2b stock solution was manifestly greater than those of the injection. Our study indicated that the IFN alpha-2b microspheres prepared in 15% of 0.89 dL/g PLGA provided a sustained drug effect for up to 21 days in rats.