A major challenge of gene therapy is to achieve highly specific transgene expression in tissues of interest with minimized off-target expression. Ultrasound in combination with microbubbles can transiently increase permeability of desired cells or tissues and thereby facilitate gene transfer. This kind of ultrasound-driven transgene expression has gained increasing attention due to its deep tissue penetration and high spatiotemporal resolution. However, successful genetic manipulation in vivo with ultrasound need to well optimize various aspects involved in this process. Ultrasound parameters, microbubble dose, and gene vectors need to be optimized for highly increased transgene expression in the cells of interest. Conversely, the potential off-target transgene expression and toxicities need to be reduced by modification of gene vectors and/or promoter sequence. This review will discuss some major strategies for enhanced specificity of the ultrasound-mediated gene transfer in vivo. Five major strategies will be discussed, including the integration of real-time imaging methods, local injection, targeted microbubbles loaded with nucleic acids, stealth nanocarriers, and cell-specific promoter. The advantages and limitations of each strategy were outlined, hoping to provide a guideline for researchers in achieving high specific ultrasound-driven gene expression.