We present a water-assisted microwave bonding (H2O-μWave bonding) process for sealing thermoplastic microfluidic devices. We use water as the bonding reagent to assist the formation of covalent bonds after surface activation and water evaporation procedures. In surface activation step, we exam UV/Ozone and O2 plasma surface treatments and their effects of the surface wettability, optical transmissivity, surface morphology, and surface chemistry on a polymethyl methacrylate (PMMA) substrate in H2O-μWave bonding. After surface activation, water evaporation step was performed to form the bonds. The bonding can be simply formed by leaving the surface-activated pair in the room environment after water application. We examined the bonding strength and coverage of PMMA by both room temperature drying and microwave annealing approaches in H2O-μWave bonding. Finally, stability and multilayer bonding were also tested to evaluate the quality of H2O-μWave bonding.