Aim: To develop and optimize a sustained release multivesicular liposome (MVL)formulation of interferon (IFN) α-2b. Methods: IFN α-2b MVL were prepared using a typical double-emulsion procedure. The sustained release effects of IFNα-2b MVL were investigated by monitoring the blood IFN α-2b concentration using an enzyme-linked immunosorbent assay test after subcutaneous administration to healthy mice. Results: IFN α-2b was successfully encapsulated in MVL with high efficiency, and the integrity of encapsulated protein was maintained. After subcutaneous injection, the MVL slowly released IFN α-2b into systemic circulation in a sustained manner. The estimated serum half-life of IFNα-2b was approximately 30 h. In addition, varying the size of the MVL preparations could further modify the in vivo release profile. Conclusion: IFN α-2b MVL may be a useful sustained release formulation in the clinical treatment of viral diseases.