Neutrophils are the most abundant non-specific immune cells and are critical in researching pathogen invasion resistance. Providing abundant cellular materials is fundamental for understanding the function and immune defense mechanisms of neutrophils. In the study, neutrophils were isolated from grass carp kidneys using a fish-tissue neutrophils isolation kit. The neutrophils were purified by the differential adherent method, and their morphology was identified by Diff’s staining and electron microscopy. The characteristic enzyme myeloperoxidase (MPO) was identified by potassium iodide and pyrro-red G staining. Using a multifunctional microplate reader, its activity was detected by the ability to express MPO and the ability to release reactive oxygen species (ROS) and nitric oxide (NO), stimulated with Phorbol 12-myristate 13-acetate (PMA). Neutrophils could also form neutrophil extracellular traps (NETs). The expression of MPO and the ability to release ROS, NO, and NETs were detected by a multifunctional enzyme plate analyzer. Survival was detected by the trypan blue staining and CCK-8. Results showed that the isolated cells had the morphological characteristics of neutrophils: they were uniform in size, with round or kidney-shaped nuclei, had type A and B particles in the cytoplasm, and indicated the characteristic sepia enzyme of MPO by staining. Purity reached (99.3±0.53)%, the number of viable cells reached (97.70±0.76)%, and the viability remained at (89.91±3.56)% after they were cultured in vitro for 24 h. By stimulation with PMA, the expression of MPO, ROS, NO, and NETs was significantly increased and positively correlated with time. The method of isolating neutrophils from grass carp was successfully established in this study, as the neutrophils showed increased vitality. Furthermore, this study provides a foundation for further research on the function and immune mechanism of fish neutrophils.