Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.