Despite their economic significance, brachyurans are relatively understudied among invertebrate groups compared to other decapods, with numerous fields remaining to be explored, including the immune response. Therefore, the aim of this study was to characterize several immune-related enzymes and activities of the Atlantic blue crab (Callinectes sapidus) in cell-free haemolymph (CFH) and in the haemocyte lysate supernatant (HLS), considering the gender factor and the suitability of measuring each activity in CFH or HLS. Data were expressed by millilitres of haemolymph or milligrams of protein to identify which of these units would be more robust and informative. For this, haemolymph from crabs of each gender was collected, and centrifuged to obtain CFH samples, whilst the cells were sonicated to get the HLS. Our results revealed that the protein concentration was higher in CFH compared to HLS. Considering milligrams of protein as units, phenoloxidase and esterase activities were lower in CFH regarding to HLS in both genders. Similarly, the lysozyme and phosphatase activities were lower in CFH compared to HLS but only in females. The values of lysozyme activity were higher in HLS of females compared to males. Studying millilitre of haemolymph as units, lysozyme activity exhibited higher activity in CFH compared to HLS in both genders, whilst phenoloxidase and esterase activities were higher in HLS, showing variations only in males. Interestingly, alkaline phosphatase activity was undetected in CFH male. Any variation was observed between the activities measured in both genders. Protease and antiprotease activities were higher and lower in CFH compared to HLS values, respectively, whilst any variation was observed between genders. Bactericidal activity against V. harveyi, V. alginolyticus and V. anguillarum, exhibited stronger values in HLS than in CFH, independently of gender, as well as against P. damselae in females. Considering gender factor, the bactericidal activity against V. alginolyticus, V. anguillarum and P. damselae was higher in HLS of females than in males. However, the bactericidal activity against V. alginolyticus and P. damselae was higher in CFH of males compared to females. Our results could contribute to gender selection in future brachyurans studies considering the variability between males and females due to their distinct biology. Additionally, we provide data regarding the baseline values of several immune-related activities, establishing the most appropriate units from the biological point of view.