A highly reproducible electrochemical biosensor, employing a five-stranded four-way junction (5S-4WJ) system through square wave voltammetry, has been successfully validated for the detection of Influenza A virus (InfA). A comprehensive assessment of its linearity, precision, accuracy, and robustness has demonstrated its compliance with FDA standards. Integration with Nucleic Acid-Based Amplification (NASBA) has showcased its selectivity for InfA, enabling the detection of InfA RNA with a standard heater set at 41 °C. This platform offers a straightforward setup well-suited for use at low-resource facilities.