Nowadays, microfluidics is attracting more and more attentions in the biological society and has provided powerful solutions for various applications. This paper reported a microfluidic strategy for aqueous sample sterilization. A well-designed small microchannel with a high hydrodynamic resistance was used to function as an in-chip pressure regulator. The pressure in the upstream microchannel was thereby elevated which made it possible to maintain a boiling-free high temperature environment for aqueous sample sterilization. A 120 °C temperature along with a pressure of 400 kPa was successfully achieved inside the chip to sterilize aqueous samples with E. coli and Staphylococcus aureus inside. This technique will find wide applications in portable cell culturing, microsurgery in wild fields, and other related micro total analysis systems.