<p>This study introduces the synthesis of amino acid-based nitrogen-doped carbon dots (N-L-Ser-CDs) from L-serine (L-Ser) and urea through a straightforward and economical one-step solid-phase pyrolysis process. The incorporation of nitrogen into the carbon dots resulted in a remarkable 27.6-fold increase in fluorescence intensity, featuring a peak emission at 405&#xa0;nm when excited at 330&#xa0;nm and a significant fluorescence quantum yield of 22.5%. These N-L-Ser-CDs displayed a specific binding affinity for Cu²⁺, leading to a pronounced fluorescence quenching effect. However, upon interaction with glutathione (GSH), the fluorescence of the N-L-Ser-CDs + Cu²⁺ complex was selectively restored. This restoration was attributed to the displacement of Cu²⁺ from the surface of the N-L-Ser-CDs due to the strong interaction between GSH and Cu²⁺. The mechanism underlying this fluorescence quenching was elucidated as an electron transfer process from the excited state of the N-L-Ser-CDs to Cu²⁺. Additionally, the sensor developed in this study exhibited a linear detection range of 0–90 µM for Cu²⁺ with a detection limit of 3 µM, and a linear detection range of 0-120 µM with a detection limit of 3 µM for GSH. By integrating the detection capabilities for both Cu²⁺ and GSH, a successful “on-off-on” fluorescent probe was developed. Most importantly, this proposed method offers simplicity, affordability, and ease of use, while also showing potential for practical GSH detection in real urine samples.</p>