<i>Kudoa septempunctata</i>, a myxozoan, has been identified as the causative agent of foodborne illnesses associated with the consumption of raw olive flounder. To develop effective control methods against this parasite, fundamental research—including viability determination, transcriptome analysis, and antigenicity assessment of <i>K. septempunctata</i> myxospores—is required. This research necessitates the purification of the parasites. Sequential trypsin digestion, followed by density gradient purification, was performed to isolate the <i>K. septempunctata</i> myxospores. Further purification was achieved through fluorescence-activated cell sorting at a concentration of 10⁶ to 10⁷ myxospores/ml. The results demonstrated that the combination of trypsin digestion and density gradient methods consistently produced approximately 40 times more viable myxospores than the density gradient method alone. Additionally, the fluorescence-activated cell sorting method enhanced the purity of the myxospores by approximately 10%. The procedures described in this study will support research (such as RNA-sequencing, proteomics, vaccine antigen preparation) aimed at developing control methods for <i>K. septempunctata</i> including the fundamental research.