The development of highly sensitive biosensors for the detection of biomolecules, such as the biomarker interleukin-8 for the early detection of oral cancer, requires optimization of sensor design. To augment the performance of an electrochemical sensor, this study used a microscale, aptamer-based electrochemical sensor for detecting botulinum neurotoxin aptamer hybridization. We first used top—down lithographic processing to define the pattern of the electrodes and then used bottom—up manufacturing to modify the surface molecular properties for reducing nonspecific binding. We systemically examined the effects of the design parameters of an aptamer-based electrochemical sensor. Specifically, five key design parameters were examined: the area of the working electrode (WE), the area of the counter electrode (CE), the separation distance between the WE and CE, the overlap length between the WE and CE, and the aptamer concentration. Through an analysis of the signal and noise generated across variations of the different parameters, the significance of each parameter in sensor performance was determined. In particular, we found that the area of the WE was the only key parameter that influenced the performance of the sensor. The output signal level increased with the area of the WE and the signal-to-noise ratio was about constant in the tested range (i.e., from 0.02 to 4 mm2).