Blood cells play an important role in animal immune system. Accurate classification and functional analysis will facilitate the understanding of immune response mechanism. In this research, hemocyte populations from Litopenaeus vannamei of 17-23 g in weight were identified through micro-observation and flow cytometry, which referred to distinguishable intracellular partical structures and measurable side scattering characteristics of cells separately. Further analyses of verifying the response hemocyte groups and their quantitative changes in early stage of bacteria or virus infection were also carried out. Four hemocyte populations of non-granular, small-granular, intermediate-granular and large-granular hemocyte could be identified by micro-observation and flow cytometry, accounting for (69.88%±4.71%)/(74.88%±3.97%), (9.04%±5.06%)/(20.75%±4.07%), (11.13%±7.02%)/(2.94%±0.72%) and (9.96%±1.31%)/(1.25%±0.58%) of the total hemocytes respectively. In both methods, non-granular hemocyte accounted for about 70%, but proportions of other 3 granular hemocytes were different depending on the method. Lipophilic tracer DID staining showed intracellular granules were covered by lipid membrane. Size similarity of granules in both intermediate-granular hemocyte and large-granular hemocyte inferred an endogenous mechanism, supporting granules were derived from golgi apparatus. The size distribution range of large-granule hemocytes was narrower and their cytoplasm was full of granules, indicating they might be at a mature stage. Further results from low intensity challenging of bacterial or virus pathogens showed that in the early time of Vibrio parahaemolyticus or WSSV infection, total cell counting reflected a similar trend of “down and up”; non-granular and small-granular hemocytes, the two major phagocytic populations, were main response groups. The low point of cell counting in V. parahaemolyticus infection experiment was observed at 24 h; but for WSSV infection, low counting period was between 36 and 48 h, which should be related to the longer time required by virus for entering cell and replicating. These support that a large number of hemocyte could be lost in a short period of time after infection. On the other hand, full of granules and showing little count fluctuation after infection, both intermediate-granular hemocyte and large-granular hemocyte might participate in immune defense through secreting bioactive molecular other than phagocytosis. Above results indicated that hemocytes populations from different crustacean species can be distinguished with similar quantitative criteria and they respond to pathogen invasion in different ways after infection.