More than the normal physiological concentration of superoxide anion radicals in and around the cell may significantly and quite often adversely modify the structure of several crucial biomolecules like DNA, proteins, and lipids that may lead to cytotoxicity and/or disease development. In this study, we generated superoxide anion radical in vitro from riboflavin under cool white fluorescent light of 800 Lux. The radical formation was confirmed by its quenching with superoxide dismutase enzyme and reduction of nitrobluetetrazolium dye. Double stranded DNA exposed to superoxide anion radical showed hyperchromicity, single strand breaks, decrease in melting temperature, and modification of thymine, adenine and guanine bases. The superoxide-modified DNA was used as antigen to detect modified-DNA reactive antibodies by immunoassay in sera of patients diagnosed for cancer of breast, lung, liver, urinary bladder and gall bladder. In competition ELISA using superoxide-modified DNA and control DNA as inhibitors, serum antibodies from cancer patients were found to recognize superoxide modified-DNA better than control DNA under identical conditions. The results of the study are significant because autoantibodies are being recognized with increasing frequency in cancer and humoral immunity is emerging as a prominent response in many different types of cancer.