<p>A novel, highly sensitive fluorometric method for D-penicillamine (D-PA) detection has been developed, addressing critical needs in therapeutic drug monitoring. D-PA, a crucial chelating agent used in treating Wilson’s disease, rheumatoid arthritis, and heavy metal poisoning, requires precise quantification due to its narrow therapeutic window and potential toxicity. The proposed method introduces an innovative sensing platform utilizing red-emissive carbon dots (R@CDs) and cobalt ions, which enables exceptional analytical performance through a sophisticated dual-quenching mechanism. The detection strategy involves cobalt ions initially enhancing the fluorescence of R@CDs. Upon D-PA addition, two synergistic quenching effects occur: competitive displacement of cobalt ions from the carbon dot surface and formation of a yellow-colored D-PA-cobalt complex, inducing an inner filter effect. Comprehensive characterization and mechanistic investigation revealed the intricate molecular interactions governing this detection process. The developed method demonstrated excellent analytical performance, exhibiting excellent linearity (R² = 0.9971) and an ultra-low limit of detection of 0.0041 µM. Rigorous validation was conducted through application to rat plasma samples, successfully quantifying D-PA in both healthy and diabetic animal models. Pharmacokinetic analysis unveiled variations in drug disposition between healthy and diabetic rats, highlighting the method’s potential for personalized therapeutic monitoring.</p>