Carbon quantum dot/carbon dot (CD) exhibiting sustained photoluminescence at longer wavelengths in aqueous solution is difficult to prepare, but has enormous potential in biomedical applications. For the first time, we report the magnesium(II) selective fluorescence enhancement of a red-light emitting anthrarufin and boric acid-derived CD in aqueous solution for direct evaluation of creatine kinase (CK) enzyme activity. The CD displayed visually detectable, intense red fluorescence only in the presence of magnesium ion (Mg²⁺) at physiological pH value when irradiated with an ultraviolet (UV) source. Concurrently, a significant increase in steady-state fluorescence intensity and fluorescence lifetime was documented. A time-dependent density functional theory (TD-DFT) analysis displayed a bathochromic shift in UV-visible (vis) absorption, and increased oscillator strength of transition resulting from the selective chelation of Mg²⁺ with β-hydroxy keto functionality on the surface of the CD. The CD-Mg²⁺ assembly was subsequently used to conceptualize the detection of CK directly through the exploration of the differential binding affinity of Mg²⁺ with adenosine triphosphate (ATP), adenosine diphophate (ADP), and CD that is otherwise not possible with commercially available kits as of today. Thus, the report delineated here usher grandeur potential of CD for biological explorations related to Mg²⁺ or ATP sensing and monitoring of Mg²⁺-dependent enzymatic activity through a clear understanding of the chemistry.