Focused ultrasound (FUS) has been widely adopted in medical and life science researches. Although various physical and biological effects of FUS have been well-documented, there is still a lack of understanding and direct evidence on the biological mechanism of therapeutic cell ablation caused by high-intensity ultrasound (HIFU) and the subsequent wound healing responses. This study develops an enclosed cell culture device that synergistically combines non-invasive FUS stimulation and real-time, on-the-fly live-cell imaging, providing an in vitro platform to explore short and long-term biological effects of ultrasound. The process, mechanism, and wound healing response of cell ablation induced by HIFU are elucidated, revealing a unique mechanism, termed ultrasound-inflicted cellular mechanolysis, that is mediated by growing subcellular cavitation air bubbles under confined contact with cells. This provides a previously unappreciated mechanism for understanding the biomechanical principles of ultrasound-based ablative therapy. A post-ablation phantom layer is also revealed that serves as a guiding cue for collective cell migration during wound healing, thereby providing a biomimetic model for studying wound healing after HIFU-inflicted damage. Together, this study provides theoretical and technological basis for advancing the understanding of the biological effects of ultrasound-based ablative therapy and inspiring clinically relevant applications in the future.