Trypanosomes are parasitic protozoan flagellates harboring in hosts' blood. Various methods have been reported to of)tain trypomastigotes from mankind and livestock to facilitate a more accurate diagnosis and to obtain purified trypomastigotes for biological, chemical, or immunological Studies Of these pathogen. However. there are so far no effective and practical methods to isolate Trypanosoma monopteri.In this study, a practical method based on Overath's method for isolating T. monopteri from blood of natural infected Monopterus albus was reported. Fish were anaesthetized with 0.2mg/mL tricaine methanesulphonate (MS222). Whole blood was obtained from the Fish caudal vein using a 2mL, heparinized syringe. Most of the erythrocytes were removed in different centrifugation speed (2.1 x 10(3) r/min, 2.5 x 10(3) r/min, 2.9 x 10(3) r/min, 3.3 x 10(3) r/min 3. 6 x 10(3) r/min) and different time (5min, 10min, 15min, 20min) with 51% Percoll reagent containing PGSS. The result showed that centrifugation at 2.9 x 10(3) r/min and 15min is best for removing erythrocytes and recovery of trypanosome. The collected suspension contained trypomastigotes, white cells, platelets and few red blood cells. To remove the remaining blood components, DEAE-52 cellulose column equilibrated with PSG was employed. The process of going through DEAE-52 cellulose column should be controlled within 1-1.5 hours. The average rate of recovery of blood trypanosomes was 88.98% and 83.80% respectively. The trypanosomes purified by this technique were morphologically normal and actively motile as checked by light microscopy and looked like condensed milk, no residual blood cells. In Conclusion, this effective and feasible technique may provide a valuable tool for further Study of parasite-host relationship, as well as the biology, biochemistry. immunology of the flagellate and prophylaxis of the trypanosomiasis. In addition, this technique has a potential diagnostic value and may provide reliable data for the Study of epidemiology of T. monopteri.