The purpose of this study was to evaluate the effect of multiple application of all-in-one dentin adhesive system on microtensile bond strength using confocal laser scanning microscope and microtensile bond strength test. Flat occlusal dentin surfaces were prepared using low-speed diamond saw. In group I, Scotchbond Multipurpose (SM) was applied by manufacturer’s recommendation. In group II, after Adper Prompt L-Pop was applied for 15s and light cured for 10s, the second coat was re-applied and light-cured. In group III, after light-curing the second layer, the third coat was re-applied and light-cured. Specimens bonded with a resin-composite were sectioned into resin-dentin stick for measuring the adhesive layer thickness by confocal laser scanning microscope and evaluating micro-tensile bond strength. The adhesive layers of three-step dentin adhesive system, 3 coats of Adper Prompt L-Pop had significantly thicker than SM, 2 coats of Adper Prompt L-Pop (p < 0.05). However, there was no significant differences in bond strengths between SM and 3 coats of Adper Prompt L-Pop (p > 0.05). And SM, 3 coats of Adper Prompt L-Pop had significantly higher than 2 coats of Adper Prompt L-Pop in bond strengths (p < 0.05).