The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.