The paper describes the preparation of monoclonal antibodies against 5-methyl-2′-deoxycytidine (5MedCyd) and its utility in the development of a competitive immunoassay system for 5MedCyd. The hybridoma was constructed by fusion of the spleen cells removed from mouse immunized with 3′-succinyl-2′-deoxycytidine keyhole-limpet hemocyanin conjugate with SP2/0-Ag14 myeloma cells. Three high titer clones that secreted anti 5MedCyd antibodies were selected. The antibodies from the three clones recognize 5MedCyd; however, small cross reactivity was observed with 5-methylcytidine and 5-methylcytosine, which possess 5-methylcytosine moiety in their structure. The clone that showed the lowest cross reactivity with these compounds was designated as 8C-4. 8C-4 antibody, which had very slight cross reactivity with the other adenosine and guanosine analogues tested in our investigation particularly the methylated ones. This monoclonal antibody was used for establishment of an immunoassay method for 5MedCyd. In this method, 8C-4 antibody was allowed to react with the free 5MedCyd in a standard solution or a urine sample in competition with 3′-succinyl-2′-deoxycytidine bovine serum albumin conjugate immobilized on microtiter plate wells. Bound antibody was quantified with alkaline phosphatase-labeled goat anti-mouse secondary antibody and p-nitrophenylphosphate substrate solution. A linear relationship with good correlation coefficient was obtained over the range 1–40μM. The specificity, reproducibility and accuracy of the proposed method was proved. There is a good correlation between the results obtained by this method and by liquid chromatography over the entire linear range. By this system, evaluation of the urinary 5MedCyd level as a biological marker for leukemic patients will be possible.