The photochemical effects of Ultra Violet-C (UV-C) radiation on the fluorochrome, acridine orange (AO) in human breast cancer cells (MCF-7) has been investigated for effective anticancer therapy. Fluorescence enhancement of AO under UV-C irradiation has been evaluated in a cell free environment using fluorescent spectral studies. Further, its photo-toxic effects were assessed using MTT assay, NBT assay and apoptotic studies through Hoechst staining and fluorescent activated cell cycle analysis. The results demonstrate that AO under UV-C exhibits enhanced fluorescence and dose dependent cytotoxicity with significant ROS production at higher doses compared to light and dark toxicity. Apoptotic studies reveal that higher exposure to UV-C of intracellular AO significantly increases the Sub-G1 fraction and morphological changes were distinct compared to the control cells. These results demonstrate that UV-C could be an ideal light delivery system for AO mediated photo-chemotherapy and can be utilized for effective anti-cancer therapy.