Carbon dots belong to the class of nanomaterials invented accidentally and are attracting a lot of attention these days. Carbon dots are non-toxic, photostable, and easy-to-synthesize nano formulations having good water-soluble properties when treated chemically by manipulating surface active groups, followed by the addition of solubilizing agents and size reduction. These are widely used in bioimaging, electrochemical sensing, targeted drug delivery, and other biomedical activities. In recent years, significant attempts have been emphasized by analysts to the detection of vitamins embedded carbon dots using biosensors. The biosensing of vitamins has become easy due to the luminescence property of carbon dots, which makes them easy to detect. Therefore, in this review, we have reported synthetic strategies and recent biosensorbased detection techniques used in the analysis of vitamin-loaded carbon dots. Even from the carbon dot’s analytical perspective, there is still a lot of research needed in the area of biosensing, bioimaging, and healthcare applications. Unique features, along with the controllable synthesis methods, will lead to a bright future in the detection and characterization of drugs using carbon dots.