Objective To investigate the clinical values of folic acid, cobalamin and homocysteine in the differential diagnosis of megaloblastic anemia (MA) and myelodysplastic syndrome (MDS of old Chinese patients. Methods 121 patients with MA and 81 patients with MDS were included in this retrospective study. Peripheral hemogram, serum level of hematopoietic material, total bilirubin and indirect bilirubin and serum level of homocysteine were compared. Results Mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, bilirubin and homocysteine level of the MA group were significantly higher than those of the MDS group (P<0.05). White blood cell count, red blood cell count, serum level of vitamin B12 and serum ferritin of the MA group were significantly lower than those of the MDS group (P<0.05). Hemoglobin, platelet count, red cell distribution width and serum level of folic acid were not significantly different between the MDS group and the MA group (P>0.05). Conclusion For patients suspected of suffering from MA or MDS, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, serum level of total/indirect bilirubin, serum ferritin are not valuable for the differential diagnosis of macrocytic anemia of old Chinese patients. Serum folic acid, vitamin B12 and homocysteine are valuable. In institutions where folic and/or vitamin B12 test is unavailable, high level of homocysteine indicates folic and/or vitamin B12 insufficiency, which indicates megaloblastic anemia is more likely the diagnosis.