Carbon dots (CDs) have been receiving increasing attention in the field of nanozymes owing to their excellent physical and chemical properties and low toxicity. However, most reported CDs are doped with metals and nitrogen elements. Few studies have investigated the mimic enzyme activity of undoped CDs. In this study, we developed a facile one-pot hydrothermal strategy to synthesize CDs using citric acid as a single carbon source (denoted as CA-CDs). Further, we found that CA-CDs possessed excellent cysteine (Cys) oxidase-mimicking activity. The CA-CDs showed typical Michaelis-Menten kinetics and better affinity for Cys than natural Cys oxidase. Based on the Cys oxidase-mimicking activity of CA-CDs, a simple and sensitive “turn-on” mode fluorescent sensor for the detection of Cys was constructed. The proposed method had a detection limit of 0.036 µM with excellent selectivity and stability and has been successfully applied to the detection of Cys in serum samples. More importantly, this research not only expands the nanozyme field of CA-CDs without doping but also provides a research foundation for the synthesis and design of carbon-based nanozymes.