Introduction: Low-energy helium-neon (He-Ne) laser beam lightis used in combination with sodium hypochlorite (Na2HOCl3) for clinical purposes. Regarding this, the present study aimed to investigate the effect of He-Ne laser (632.8 nm) and sodium hypochlorite on the calf thymus double-stranded deoxyribonucleic acid (ctdsDNA) molecule. Materials and Methods: For the purpose of the study, ctdsDNA solutions (30µg/ml) were exposed to He-Ne laser (632.8 nm) light in the absence and presence of different concentrations of sodium hypochlorite for up to 60 sec. The levels of nucleic acids released as uncontaminated and contaminated proteins were considered as the markers of DNA damage in terms of hypochromasia (i.e., DNA strand breakage) and hyperchromasia (i.e., of DNA strands separation). Results: The mean concentration of nucleic acids insignificantly (P > 0.05) decreased after exposure to laser light irradiation (hypochromic effect). Furthermore, laser irradiation insignificantly and inconsistency protected the ctdsDNA molecules from the effect of sodium hypochlorite. Sodium hypochlorite at concentrations of 1 and 3 mmol reduced the levels of the nucleic acids released from contaminated protein by 29.2% and 78.3% of the pre-incubated levels (hyporchromasia effect). The He-Ne laser (632.8 nm) irradiation induced hypochromic effect on the uncontaminated and contaminated proteins, while sodium hypochlorite induced a remarkable hyperchromic effect at higher concentrations. Conclusion: As the finding indicated, a short time He-Ne laser light (632.8 nm) irradiation exerted minor significant effect on the ctdsDNA molecule. This laser light did not interact with sodium hypochlorite as a synergistic combination against the ctdsDNA molecule.