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Molecular imprinting has emerged as an important and practical technology to create economical and stable synthetic mimics of antibodies and enzymes. It has already found a variety of important applications, such as affinity separation, chemical/biological sensing, disease diagnostics, proteomics, bioimaging, controlled drug release, and catalysis. In the past decade, significant breakthroughs have been made in non-covalently imprinted polymers, from their synthesis through to their applications. In terms of synthesis, quite a few versatile and facile imprinting approaches for preparing MIPs have been invented, which have effectively solved some key issues in molecular imprinting. Additionally, important applications in several areas, such as sensors, proteomics and bioimaging, have been well demonstrated. In this review, we critically and comprehensively survey key recent advances made in the preparation of non-covalently imprinted polymers and their important applications. We focus on the state-of-art of this technology from three different perspectives: fundamentals, synthetic strategies, and applications. We first provide a fundamental basis for molecular imprinting technologies that have been developed, which is extremely helpful for establishing a sound understanding of the challenges in molecular imprinting. Then, we discuss in particular the major breakthroughs within the last ten years (2014–2024), with emphasis on new imprinting approaches, what strengths the breakthroughs can provide, and which new applications the properties of the prepared non-covalently imprinted polymers are fit for.