A new ratiometric fluorescence strategy for sensitive and selective detection of cobalt ions (Co²⁺) and topotecan (TOP) is proposed. The dual-probe system consists of red-emissive nitrogen and sulphur doped carbon dots (R-NS@CDs) and TOP. For Co²⁺ detection, the fluorescence of R-NS@CDs at 680 nm is enhanced upon addition of Co²⁺, while complexed TOP emission peak at 545 nm remains constant. This enables ratiometric detection of Co²⁺ over a range of 5.0–160.0 ng mL⁻¹. The mechanism of R-NS@CDs fluorescence enhancement by Co²⁺ is elucidated using FTIR, fluorescence spectroscopy, zeta potential measurements, and TEM imaging. For the detection of TOP, a ratiometric probe system comprising R-NS@CDs and Co²⁺ was utilized. TOP forms a complex with Co²⁺ bounded to R-NS@CDs, quenching the R-NS@CDs-Co²⁺ fluorescence and simultaneously the native TOP fluorescence is enhanced. This allows ratiometric quantification of TOP from 1.0-90.0 ng mL⁻¹. The method provides high selectivity and low detection limits of 1.51 ng mL⁻¹ for Co²⁺ and 0.37 ng mL⁻¹ for TOP. Practical applicability is demonstrated through selective detection of Co²⁺ in environmental water samples and TOP in real plasma samples. The built-in self-calibration enabled by dual analyte modulation of R-NS@CDs makes this a simple and powerful analytical approach.