A phenylboric acid functionalized carbon dot (2-FPBA-CD) for rapid fluorescent sensing of glucose in blood was synthesized by simply mixing N, S-doped carbon dots (CDs) with phenylboric acid at room temperature. At pH 7.4, the response of 2-FPBA-CD to glucose could reach equilibrium in a very short time (10 min), with a wide responsive linear range of 19.70 μM to 2.54 mM, which can be applied to the detection of glucose in serum. The mechanism studies showed that the layered carbon film of 2-FPBA-CD aggregated after adding glucose, thereby leading to the fluorescence quenching of 2-FPBA-CD.