A new optical sensing microplate was developed for rapidly determining Gemifloxacin Mesylate in its pure form, pharmaceutical preparations, and human plasma using new Tb(III)-complex with 3-allyl-2-hydroxybenzohydrazide (AZ) as a probe. The interaction of Gemifloxacin mesylate with the Tb (III)- (AZ)2 complex is based on the luminescence enhancement. The metal to ligand ratio of the complex is 1:2 as determined by a Job plot method. The calibration graph was linear over the range of 0.15–10 µg/mL, with a limit of quantification (LOQ) of 0.15 µg/mL and a detection limit (LOD) of 0.05 µg/mL. The thermodynamic analysis for the interaction GMF with Tb (III)–(AZ)2 complex was evaluated. The thermodynamic analysis showed that the interaction between the Tb (III)–(AZ)2 complex and GMF was spontaneous with negative ∆G. The established approach has been used successfully to the determination of drugs in pharmaceutical formulations and spiked plasma samples with good recovery percent and RSD less than five percent.