A simple and rapid ratiometric fluorescent sensing system for d-penicillamine (D-PA) determination is developed based on yellow carbon dots (Y-CDs) combined with thiochrome (oxVB1) for the first time. The oxidization of thiamine (VB1) can be catalyzed by Alkaline-hydrolyzed artemisinin (a-ART) to form oxVB1, which leads to the occurrence of fluorescence emission peak at 466 nm. Furthermore, the oxidation reaction between a-ART and VB1 could be inhibited by D-PA, and accompanied with the decrease of fluorescence at 466 nm. However, the fluorescence peak of Y-CDs as an internal reference at 566 nm was almost unchanged. The ratiometric signal changes contributed to a robust and sensitive D-PA sensing. Under the optimal condition, a good linear response for the D-PA detection was obtained in the ranges of 0.5–50 μM with a detection limit of 0.33 μM. In addition, Y-CDs and thiochrome-based sensing system was applied to D-PA determination in real samples and obtained acceptable results.Graphical abstract We developed a new carbon dots/thiochrome fluorescent nanoprobe for ratiometric fluorescence sensing of d-penicillamine.