The extracellular matrix (ECM) plays a pivotal role in regulating cellular behavior and driving tissue regeneration. Its unique structural characteristics and bioactivity not only provide physical support for cell growth, but also orchestrate tissue repair and functional reconstruction through multiple signaling pathways. This review systematically synthesizes preparation strategies for natural and engineered ECM materials from the perspective of ECM-mediated tissue regeneration mechanisms, with particular emphasis on recent advances in component preservation, structural biomimicry, and functional optimization. Furthermore, it delves into the application potential of cutting-edge technologies—including artificial intelligence, flexible electronics, and organoids—in ECM engineering, while critically analyzing the standardization and safety challenges hindering clinical translation. This article aims to provide a theoretical foundation and reference for constructing next-generation ECM-based regenerative medicine platforms.