Monitoring small neurochemicals such as serotonin is crucial for better understanding brain function, neurodegenerative and psychiatric disorders. However, detecting small molecules in complex media remains challenging due to the impact of non-specific binding, especially at low target concentrations. This master’s thesis explores the integration of aptamer-based recognition with focal molography, an optical biosensing technique that inherently filters non-specific binding, to enable serotonin detection at physiologically relevant nanomolar concentrations. To overcome the challenges posed by serotonin’s small size and low molecular mass (<200 Da), a competitive binding assay (CBA) was implemented. Two assay formats were investigated: direct CBA, in which serotonin competes with a mediator strand for aptamer binding at the sensor surface, and inverse CBA, in which serotonin is pre-incubated with aptamers in solution before surface hybridization.