The pace of change in chemical and biological research enabled by improved detection systems demands fundamental liquid handling and sample preparation changes. The acoustic droplet ejection (ADE)-based liquid handling method has the advantages of improving precision and data reproducibility, reducing costs, hands-on time, and eliminating waste. ADE gradually replaced traditional aspiration-and-dispense liquid-handling robots in applications such as synthetic biology, genotyping, personalized medicine, and next-generation sequencing. This review emphatically introduces the setup of the ADE system and the critical technologies of each part, including acoustic droplet generation, optimized design of the source fluid wells, droplet coalescence, and power control. The advantages and disadvantages of these technologies are discussed, and the future development of acoustic droplet ejection technology is also predicted.