With the development of molecular biology, genetic engineering of microorganisms has been increasingly applied in microbial breeding, clinical medicine and environmental bioremediation. However delivery of DNA into the cell can be one bottleneck in efficient genetic engineering. Ultrasound-mediated DNA delivery into microbial cells, with a number of advantages that include in situ operation, spatial scalability, non-invasiveness, high-throughput and low consumable costs, has been under rapid development. Ultrasound can produce a variety of nonthermal effects via acoustic cavitation, and the cavitation bubbles generated during this process can cause transient cell membrane permeability. In this review the basic principles of the ultrasound-based DNA delivery were discussed and its current applications in prokaryotes were summarized. Furthermore, we discussed the advantages and challenges of the technique, using the transformation of Gram-positive bacteria in our laboratory as an example.