Hybrid spectroelectrochemical sensors (HSESs) have been introduced in recent years to improve the analytical performance of chemical sensors. The development of these methods is still a serious challenge due to the lack of efficient electrochemical stimulation strategy. In this research, we have reported the development of an HSES based on the electrochemical preconcentration for the quantification of ampyra (AMP), as a model analyte. In this method, the preconcentration of AMP occurred at transparent FTO/AgNPs substrate when the potential was applied as an electrochemical stimulation. Consequently, the preconcentrated AMP was determined by recording the absorbance of the substrate at 320 nm. The formation of an electrodeposited layer onto the FTO/AgNPs surface was characterized using field emission scanning electron microscope (FESEM), fluorescence microscopy, X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), chronoamperometry, and electrochemical impedance spectroscopy (EIS) techniques. FTO/AgNPs modified electrode respond to AMP in the linear range of 14.85 to 1461 μmol L−1 with a detection limit of 5.77 μmol L−1, which exhibits sensitivity levels sufficient for determining the analyte in real pharmaceutical samples. Moreover, the developed HSES showed high selectivity for AMP compared to other interfering compounds, such as pyridine and acetaminophen. This analytical strategy will open up a new door toward the fabrication of applicable HSESs.