Background <p>This study aimed to evaluate the immediate and 6-month dentin micro-tensile bond strength (µTBS) of three universal adhesives applied using different double-layer application techniques.</p> Methods <p>Sixty-five human molar teeth were divided into 13 groups (<i>n</i> = 5) based on adhesive systems and application procedures. Three universal adhesives (G-Premio Bond, GC Corp.; GP, OptiBond Universal, Kerr; OB and Single Bond Universal, 3&#xa0;M ESPE; SB) and a two-step self-etch adhesive (Clearfil SE Bond, Kuraray Noritake Dental; CSE) were tested. Universal adhesives were applied according to the manufacturer’s instructions-(SL-MI) or three different double-layer application procedures: (1) the first adhesive layer was applied without either air thinning or light curing-(DL1), (2) air thinning without light curing the first layer-(DL2), (3) with air thinning and polymerisation each layer-(DL3). The two-step self-etch group was applied according to the manufacturer’s instructions. µTBS was measured immediately, after 3 months and 6 months of water storage. Statistical analyses were conducted using Three-Way ANOVA (<i>p</i>&lt; 0.05).</p> Results <p>For the immediate and 3-month water storage of SB and GP, the highest µTBS values were observed in Group DL2, while for OB, Group DL3 exhibited the highest µTBS values (<i>p</i>&lt; 0.05). After 6 months of water storage, differences among the application procedures were not significant for GP and OB (<i>p</i>&gt; 0.05). The two-step self-etch system demonstrated higher µTBS than all universal adhesive systems at all test periods (<i>p</i>&lt; 0.05).</p> Conclusion <p>The application of universal adhesives in two layers, particularly by thinning the first layer with air and polymerising after the second layer, positively affects immediate and 3-month µTBS. However, the benefits of double-layer application on 6-month µTBS appear to be material dependent.</p>