This work presents a simple yet selective fluorometric protocol for the quantification of vancomycin, an important antibiotic for treating infections caused by Gram-positive bacteria. A novel ratiometric fluorometric method for the determination of vancomycin is developed based on dual emissive carbon dots (DECDs) with emission at 382 nm and 570 nm in combination with Co²⁺ ions. Upon addition of Co²⁺ions, the fluorescence at 382 nm of DECDs is enhanced while emission at 570 nm remains constant. In the presence of vancomycin, it complexes with Co²⁺ leading to quenching of the 382 nm fluorescence due to strong binding with Co²⁺ in the Co@DECDs system. The DECDs are fully characterized by TEM and different spectroscopic techniques. The proposed ratiometric method is based on measuring fluorescence ratio (F₅₇₀/F₃₈₂) against vancomycin concentration and the method exhibits a good linearity range from 0.0 to 120.0 ng mL⁻¹ with a low limit of detection (S/N = 3) of 0.31 ng mL⁻¹. The method shows good selectivity with minimal interference from potential interfering species. This ratiometric fluorometric approach provides a promising tool for sensitive and specific vancomycin detection in clinical applications.