<p>Glycine (Gly) was employed as a primary precursor to prepare two types of carbon dots (CDs): glycine-derived nitrogen-doped CDs (Gly-NCDs) and glycine-derived nitrogen/chlorine co-doped CDs (Gly-NClCDs). The latter were obtained by introducing diethylenetriamine and concentrated hydrochloric acid during the synthesis process. The structural and optical properties of the two types of CDs were systematically investigated and compared, revealing that their distinct photoluminescence behaviors likely originate from differences in surface states and elemental doping. These findings provide practical insights into regulating quantum yield, optical characteristics, and particle size without resorting to complex synthetic procedures or expensive reagents. Riboflavin (VB₂) is an essential micronutrient involved in various physiological processes, and its rapid and cost-effective determination is of practical importance. Gly-NCDs exhibited good selectivity and a wide linear response range for VB₂ detection, enabling ratiometric fluorescence sensing over a concentration range 0.50‒35.70 µM with a detection limit of 1.47 µM. To enhance practical applicability, a smartphone-based sensing platform was further developed for portable and real-time VB₂ detection under both visible and UV illumination. This platform achieved detection ranges of 5.00‒45.00 µM, with detection limits of 1.76 µM and 1.36 µM, respectively. The proposed method was successfully validated by determining VB₂ in pharmaceutical formulations and human serum samples, demonstrating acceptable accuracy and reproducibility. Overall, this work highlights the potential of glycine-derived CDs as low-cost, scalable, and portable nanoprobes for applications in drug quality control, environmental monitoring, and point-of-care analysis.</p> Graphical Abstract <p></p>