Antibodies are important tools in the study of protein function and diagnostic tests. However, traditional antiserum preparation requires a time-consuming immunization protocol and subsequent purification of polyclonal antibodies. In this study, a rapid and efficient method for polyclonal antibody preparation has been developed. Juxtanodin (JN) and silent information regulator-2 (Sirt2), both of which are oligodendrocyte-specific proteins, were used for antibody preparation. The N-terminal 170 amino acids of JN (JN170) and amino acids 231-351 of Sirt2 (Sirt2-121) were expressed as GST-tagged proteins from a pET-41a(+) vector in E. coli strain BL21 (DE3) cells. The fusion proteins were purified and used to immunize rabbits following both a traditional protocol, in which antigen was presented biweekly, and a modified rapid protocol, in which the immunization on day 1 was boosted on days 5 and 28. ELISA, Western blot analysis and immunofluorescent staining showed that antibodies produced via the rapid protocol could recognize these two oligodendrocyte-specific proteins in vitro and in the rat central nervous system (CNS), respectively, similar to those produced with the traditional protocol. Thus, our study provides a novel rapid method to prepare high specificity antibodies via a modified immunization protocol and subsequent antibody purification.