Fragmentation of DNA into short fragments is of great importance for detecting and studying DNAs. Current microfluidic methods of DNA fragmentation are either inefficient for generating small fragments or rely on microbubbles. Here, we report a DNA fragmentation method in a 3D-printed microfluidic device, which allows efficient continuous flow fragmentation of genomic DNAs without the need for microbubbles. This method is enabled by localized acoustic streaming induced by a single vibrating sharp-tip. Genomic DNAs were fragmented into 700 to 3000 bp fragments with a low power consumption of ~140 mW. The system demonstrated successful fragmentation under a wide range of flow rates from 1 to 50 μL/min without the need for air bubbles. Finally, the utility of the continuous DNA fragmentation method was demonstrated to accelerate the DNA hybridization process for biosensing. Due to the small footprint, continuous flow and bubble-free operation, and high fragmentation efficiency, this method demonstrated great potential for coupling with other functional microfluidic units to achieve an integrated DNA analysis platform.