as 0D and discrete, quasispherical nanoparticles with the characteristic size of less than 10 nm in shape. In the past few decades, many researchers have been intrigued by overwhelming application of CDs in fields other than solar photovoltaics, [23][24][25] semiconductor photocatalysis, [26] or light-emitting materials, [27][28][29] like in bioimaging, [30][31][32][33] drug delivery/ release, [34][35][36] theranostics, [37] and biosensing/bioassay. [38][39][40] Albeit with biological imaging agents in vitro and in vivo, heavy metals as the essential elements for the core materials of available high-performance semiconductor quantum dots (QDs) exist substantial toxicity, carcinogenic effects, and environmental hazard, even at low levels. [41][42][43][44][45] Historically, boasting the natural virtues of fascinating optical properties (e.g., up/down-conversion and excitation-dependent fluorescence emission, high fluorescence quantum yields (QY), and long fluorescence lifetime), eco-friendliness, high waterdispersible peculiarity, easy surface-functionality, low cost, highly abundant, and inexpensive raw materials, noninvasiveness, low cytotoxicity, and good biocompatibility afford CDs suitable as the elegant alternatives for toxic QDs containing heavy metals. [46][47][48][49] On the other hand, the CDs have excellent photostability against photobleaching and photoblinking as compared with the conventional organic dyes (typically fluorescein and rhodamine B). [50] In particular, multicolor CDs exhibit tunable photoluminescence by exciting the identical CDs, allowing for multimodal biophotosensitizer in biological field without interference from background autofluorescence. In this review, we aim to systematically offer a summary of preparing multicolor CDs and its various biomedical applications, mainly including for bioimaging, drug delivery/release, theranostics, and biosensing/bioassay, highlighting the complete horizons of multiplexed quantitative detection, high-resolution fluorescence imaging, and long-term, real-time monitoring of multicolor CDs.Multicolor carbon dots (CDs) as an emerging subclass of carbonaceous nanomaterials have inspired intensive attention due to the fascinating fluorogenic properties of quantum dots, exhibiting great potential applications in the biomedical field. In some cases, reported CDs with blue or green fluorescence are not desirable for further biological applications owing to the conflicting background autofluorescence, low penetration, and relatively large damage to biological tissue. However, multicolor CDs that can work in the longer wavelength region are being developed to address this issue by overcoming the autofluorescence from the cellular components (usually in the blue and green range). In this review, the development of multicolor CDs is described comprehensively, including for red-emissive or NIR-emissive CDs. Additionally, the preparation methods of multicolor CDs are summarized. Moreover, the factors affecting the luminescence of multicolor CDs are discussed in de...