AbstractAdeno‐associated virus (AAV)‐based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)‐triggered on‐demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge‐based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W‐MPs) are fabricated. Since W‐MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly‐l‐lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W‐MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US‐triggered AAV release system expands methodological possibilities in gene therapy.