Multicharged cyclodextrin (CD) supramolecular assemblies, including those based on positively/negatively charged modified mono-6-deoxy-CDs, per-6-deoxy-CDs, and random 2,3,6-deoxy-CDs, as well as parent CDs binding positively/negatively charged guests, have been extensively applied in chemistry, materials science, medicine, biological science, catalysis, and other fields. In this review, we primarily focus on summarizing the recent advances in positively/negatively charged CDs and parent CDs encapsulating positively/negatively charged guests, especially the construction process of supramolecular assemblies and their applications. Compared with uncharged CDs, multicharged CDs display remarkably high antiviral and antibacterial activity as well as efficient protein fibrosis inhibition. Meanwhile, charged CDs can interact with oppositely charged dyes, drugs, polymers, and biomacromolecules to achieve effective encapsulation and aggregation. Consequently, multicharged CD supramolecular assemblies show great advantages in improving drug-delivery efficiency, the luminescence properties of materials, molecular recognition and imaging, and the toughness of supramolecular hydrogels, in addition to enabling the construction of multistimuli-responsive assemblies. These features are anticipated to not only promote the development of CD-based supramolecular chemistry but also contribute to the rapid exploitation of these assemblies in diverse interdisciplinary applications.