Objective: The important mission in modern science of caries prevention is identification and providing the necessary actions for caries prevention to patients with an elevated risk of caries. The aim of this study was evaluation of CO2 laser irradiation effect on enamel microhardness after incipient caries creation. Material and methods: In this in vitro-experimental study, for evaluation of enamel microhardness 30 teeth after disinfection process were divided randomly into three groups A, B, C [n=10]: A] Control [normal saline] B] Immersed in cariogenic solution for 1 month C] Immersed in cariogenic solution for 1 month + CO2 laser [10.6μm, 10Hz, 0.5W, 20s, beam diameter 0.2 mm]. Data analyzing was used by 16 SPSS software. Parametric one-way ANOVA and Tukey were used for surface microhardness at 0.05% significance level. Results: According to one-way ANOVA parametric test, there was a significant difference between three groups [p=0.047]. In the following, results of Tukey test showed that there was a significant statistical difference between the microhardness of control and other groups [P=0.038]. On the other hand, there wasn’t statistical difference between A, C and B, C group means [P>0.05]. Conclusion: These study findings showed that CO2 laser irradiation on enamel surface with incipient caries had no significant effect on surface microhardness enhancement.