Polysaccharides from seaweeds are promising biomolecules that could sustainably improve the health status of cultured fish species. In this study, water-soluble polysaccharides extracted from Chaetomorpha antennina were investigated to determine their physicochemical characteristics and biological activities. C. antennina water-soluble polysaccharides (CAWP) comprised mainly of carbohydrate (69.0 ± 2.3 %), sulphate (12.6 ± 0.5 %), uronic acid (8.8 ± 0.1 %) and protein (3.5 ± 0.2 %). High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) analysis revealed that CAWP contained mostly arabinose (17.1 ± 2.3 mg/L), galactose (9.3 ± 1.0 mg/L), glucose (5.1 ± 0.6 mg/L), xylose/mannose (1.4 ± 0.6 mg/L) and a small proportion of fucose (0.3 ± 0.1 mg/L). CAWP, with intrinsic viscosity of 0.66 dL/g and estimated molecular weight of 318 kDa, were non-toxic to Carp leukocyte culture (CLC) cell line at concentrations ≤25 μg/mL. CAWP at concentrations of 12.5 and 25 μg/mL stimulated CLC to release significant amount of nitric oxide (NO), reactive oxygen species (ROS) and augmented phagocytic activity. Intracellular antioxidant activities indicated that CAWP did not induce oxidative damage in CLC but could improve superoxide dismutase activity (SOD), total glutathione (tGSH) and decrease malondialdehyde (MDA) level. The findings from our study provide a foundation for the applicability of water-soluble polysaccharides from C. antennina in the aquaculture industry as a potent immunomodulatory agent.