Bond strength of adhesive layer can absorb unwanted stresses of polymerization shrinkage in composite resin restorations; increased microshear bond strength can prevent failure of restoration materials, the purpose of this study was to evaluate the effect of adhesive layers on microshear bond strength of nanocomposite resin to dentin. Two different types of adhesive systems: universal adhesive (ExciTE) and newly developed adhesive (Nano-Bond), and one type of light-cured resin restorative material (Nanocomposite resin) were used in this study. The occlusal surfaces of extracted human molar teeth were ground perpendicular to the long axis of each tooth to expose a flat dentin surface. The adhesives were applied on dentin surfaces (single application or double application). Nanocomposite resin was then placed and light cured for 40 seconds. After 24 hours of immersion in water at 37°C, then subjected to thermocycling before testing, a microshear bond test was carried out. The data were analyzed by a two-way ANOVA. For comparison between groups, Tukey’s post-hoc test was used. The mean bond strengths of ExciTE and Nano-Bond adhesives with a single application were 8.8 and 16.6 MPa, respectively. The mean bond strengths of ExciTE and Nano-Bond adhesives with double application were 13.2 and 21.8MPa, respectively. There were no statistically significant differences in microshear bond strengths between the single application of Nano-Bond and the double application of ExciTE adhesives. Microshear bond strength increased significantly as the applied adhesive layer was doubled.