Selegiline, a selective monoamine oxidase-B inhibitor used in Parkinson's disease treatment, requires accurate detection for therapeutic drug monitoring and optimal dosing. This study presents a novel fluorometric method for selegiline detection based on azide-modified carbon dots that selectively interact with selegiline's terminal alkyne group through copper-catalyzed azide-alkyne cycloaddition (CuAAC). This specific click chemistry reaction results in fluorescence quenching of the carbon dots. The detection mechanism and comprehensive characterization of the azide-modified carbon dots are thoroughly discussed, including their synthesis, optical properties, and interaction behavior with selegiline. The method exhibits excellent analytical performance with good linearity across a wide concentration range of (0.1–80.0 ng/mL) and demonstrates a low limit of detection of 0.041 ng/mL. A selective extraction methodology employing FMOC derivatization coupled with mixed-mode cation exchange (MCX) solid-phase extraction was developed to eliminate amine metabolites and biological interferents while preserving selegiline's tertiary amine structure for selective retention and purification. The method shows high selectivity against interferents and achieves excellent percentage recovery values reaching (96.0–97.0 %) when applied to urine samples. Most importantly, the method was successfully applied to urine samples from Parkinsonism patients, providing a non-invasive route for selegiline determination that offers significant advantages over traditional invasive sampling methods.