MXenes, primarily composed of two-dimensional (2D) transition metal carbides/nitrides, have emerged as a research hot spot, particularly in the field of biomedicine, owing to their notable physicochemical properties, functional termini, and intriguing biocompatibilities. However, they suffer from smaller specific surface areas, poor mechanical properties, and a lack of dimensional control. Previous studies have indicated that constructing 2D MXene nanosheets into a three-dimensional (3D) structure can effectively reduce the level of re-aggregation, which can provide a larger specific surface area and stronger physical structural support for various applications. This work provides a systematic and focused review of the latest developments in 3D-assembled MXene for biomedical applications. This review first presents a comprehensive summary of the widely employed strategies for manufacturing 3D-assembled MXene architectures, such as metal ion-induced assembly, metal-organic framework-assisted assembly, carbon materials-assisted assembly, and polymer-assisted assembly. Furthermore, research attention is also directed toward exploring the structure-property relationships of 3D-assembled MXenes for diagnostic applications, i.e., biosensing and bioimaging, and therapeutic applications, i.e., drug delivery and tumor therapy. Finally, opportunities and challenges of the 3D-assembled MXenes have also been outlined. We strongly expect that 3D-assembled MXenes can offer significant benefits in the context of clinical translations for theranostic applications.