<p>A dual-mode nanoprobe has been presented for the quantitative determination of cefuroxime based on fluorescence and room-temperature phosphorescence (RTP) responses. Carbon dots (CDs) exhibiting stable dual emission were synthesized from dicyandiamide and tartaric acid as carbon sources. In the presence of cefuroxime, both fluorescence and RTP emissions of the CDs were selectively quenched, which were attributed to hydrogen-bonding interactions and photoinduced electron transfer. The dual-mode response enabled the sensitive detection of cefuroxime fluorescence limits of detection (LOD) of 8.7 µΜ (in water) and 7.76 µM (in plasma), and RTP LODs of 6.9 µM (in water) and 7.50 µM (in plasma), respectively. The nanoprobe showed satisfactory selectivity toward cefuroxime in the presence of common interferents and was successfully validated in plasma, with recoveries of 97–108% and relative standard deviations below 3.2% across the therapeutic concentration range. These results demonstrated a dual-mode optical platform suitable for therapeutic drug monitoring of cefuroxime.</p> Graphical Abstract <p></p>