Functional molecular markers have been widely used in the study of germ cell development of human and other model organisms, whereas less applications were in fishes. In this study, RNA Pol II CTD (S5) was used as a functional molecular marker for immunofluorescence analysis in germ cell nuclei of common carp, and the related technical parameters were further explored. The results showed that this method could detect the characteristics of mitosis phase and chromosomal immunofluorescence signals, which were related to transcriptional active sites during the development stage of fish germ cells. In addition, the quality of immunofluorescence signals were affected by the critical experimental procedures including chromosome preparation and washing specimens. This study not only provides important molecular markers served as immunoprobe for the study of germ cell development in fishes, but also provides a new detection strategy for fish genetics and breeding.