A technical bottleneck to the broadening of applications of glass nanofluidic chips is bonding, due to the strict conditions, especially the extremely high temperatures (~1000 °C) and the high vacuum required in the current glass-to-glass fusion bonding method. Herein, we report a strong, nanostructure-friendly, and high pressure-resistant bonding method, performed at room temperature (RT, ~25 °C) for glass nanofluidic chips, using a one-step surface activation process with an O(2)/CF(4) gas mixture plasma treatment. The developed RT bonding method is believed to be able to conquer the technical bottleneck in bonding in nanofluidic fields.