Background: Lamotrigine is widely used in the management of partial epilepsy, generalized tonic-clonic epilepsy and Lenox-Gastut syndrome and an add-on therapy in the treatment of complex and simple partial seizures and secondarily generalized tonic-clonic seizures resistant to multiple drug therapy. Methods: In the current study, a fluorometric nanoprobe based on metal–organic frameworks (MOF) was designed for the determination of lamotrigine in exhaled breath condensate (EBC). The MOF nanoprobe consisted of Tb3+ ions as metal part and dimethylformamide (DMF) and 1,10-phenanthroline (Phen) as organic parts of nanoprobe. Results: The used probe shows a week fluorescence in alkaline media owing to an energy transfer from nitrogen groups of DMF and Phen on carbonyl group of DMF as an antenna for Tb3+ luminescence. However, its fluorescence is enhanced in acidic conditions by protonation of DMF nitrogen atoms and Phen and deactivation of energy transfer pathways of nitrogen groups to carbonyl group. Lamotrigine addition to this fluorescent system leads to quenching in the fluorescence intensity due to reactivation of the above mentioned energy transfer pathways resulting in competitive interaction with H+ ions. Moreover, the inner filter effect (IFE) of lamotrigine on DMF–Tb–Phen MOF NPs is considered as another reason for the observed quenching in the fluorescence of DMF–Tb–Phen MOF NPs. The intensity of the fluorescence was recorded at λem = 545 nm and the difference between fluorescence signal in the absence and presence of lamotrigine was the analytical response. The factors affected on experimental conditions were optimized utilizing a multivariate optimization technique. The validation of nanoprobe response to lamotrigine gives a linear relationship in the range of 0.05 to 2.0 µg⋅mL‒1 with a detection limit of 11.0 ng⋅mL‒1 for lamotrigine. Conclusion: The developed method reveals good repeatability and selectivity for lamotrigine in real samples.