Abstract: Being rich in a large number of hemocytes, the gills of crustacean play an important role in the regulation of hemocyte homeostasis and immune defense. Molecular markers to distinguish gill cells from hemocytes are required for the study of hemocytes in gill. In this study, we compared the protein profiles of gill cells and hemocytes of crayfish Cherax quadricarinatus using SDS-PAGE electrophoresis. Eight candidate proteins were identified from tissue-specific bands by LC-MS/MS. The expression of these candidate proteins in gill and hemocytes were analyzed by quantitative real-time fluorescence PCR (qRT-PCR). The transcription of vacuolar-H⁺-ATPase subunit C (V-H⁺-ATPase) in gill was 53.3 folds of that in hemocytes. Whereas, the transcription of alpha-2-macroglobulin (α2M) and vitelline membrane outer layer protein I-like protein (VMO-Ia) in hemocytes was 10.3 folds and 10.9 folds of that in gills, respectively. Western blot and immunofluorescence analysis confirmed that V-H⁺-ATPase and VMO-Ia proteins were specifically expressed in gill cells and hemocytes, respectively. Therefore, -H⁺-ATPase and VMO-Ia can be used as molecular markers to distinguish crayfish gill cells from hemocytes and may also provide a reference for the study of gill hemocytes in other decapod crustaceans.