Microglia are the primary resident immune cells of the retina and are involved in the pathogenesis of various retinal diseases. In this study, we optimized experimental conditions to isolate microglia from canine retinas and characterized ex vivo their immunophenotype and function using flow cytometry (FACS). The most suitable protocol included a mechanical dissociation of the retina and an enzymatic digestion using DNAse and collagenase. Extraction was carried out by density gradient centrifugation, and retinal microglia accumulated on distinct interfaces of 1.072 and 1.088 g/mL of a Percoll gradient. Immunophenotypical characterization was performed with monoclonal antibodies CD11b, CD11c, CD18, CD45, CD44, B7-1 (CD80), B7-2 (CD86), CD1c, ICAM-1 (CD54), CD14, MHCI, MHCII, CD68, CD3, CD4, CD8α, and CD21. The most prevalent microglia population in the normal canine retina is CD11b(high)CD45(low). Functionally, retinal microglia exhibited phagocytosis and reactive oxygen species (ROS) generation activities. To conclude, ex vivo examinations of retinal microglia are feasible and possibly reflect the in vivo conditions, avoiding artifacts observed in tissue culture. The established method will be relevant to examine microglia from diseased canine retinas in order to elucidate their roles in degenerative processes.