Here, we report the characterization of a four-way junction (4J) universal electrochemical sensor that consists of an electrode-immobilized DNA stem-loop (SL) probe and two adaptor strands (m and f). The “m” adaptor strand was labeled with a methylene blue (MeB) redox marker. The adaptor strands hybridize to the DNA SL probe and target nucleic acid to form a 4J structure. In this orientation the MeB is in close proximity to the electrode’s surface thus enabling its reduction and subsequent electrochemical signal. The experimental timing for immobilization of the DNA SL probe and hybridization with adaptor strands and miRNA-122 was investigated. In addition, the density of the DNA SL probe coverage was evaluated using chronocoulometry to determine the ideal concentration of immobilized DNA SL probe. The sensor performance was investigated using cyclic voltammetry (CV), alternating current voltammetry (ACV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). Specifically, a single parameter from each voltammetric technique was modified to evaluate the sensor’s response. The DNA SL probe demonstrated an efficient immobilization after 30 minutes and optimal hybridization occurred at 1.5 hours. The 4J sensor was characterized with ACV at a frequency range from 1 to 5000 Hz and the current increased from 1 to 600 Hz where the threshold of the sensor was reached, followed by a drastic decrease in signal at higher frequencies. The 4J sensor was also characterized with SWV at frequencies ranging from 1 to 5000 Hz and exhibited an increase in signal from 1 to 1500 Hz, where the signal stabilized until reaching the threshold frequency of 4000 Hz, where the current then began to decrease. CV was used to evaluate the sensor’s performance at scan rates ranging from 0.1 to 800 V/s. The peak separation increased at faster scan rates (529 mV) and the signal was saturated after 500 V/s. Finally, the sensor performance was monitored using DPV at pulse widths ranging from 1 to 100 ms.  The current was highest at short pulse widths (1 ms) and the current decreased rapidly as the pulse width was increased. Amplification of the 4J sensor was achieved by modifying a specific parameter for each electrochemical technique which can provide insight into the sensor performance to facilitate lower limits of detection of nucleic acids.