In recent years, the rapid detection of viral pathogens has become critical for effective diagnostics and infection control, especially during pandemics. Traditional diagnostic methods, such as polymerase chain reaction (PCR) and antigen-based assays, have limitations related to equipment complexity, processing time, and sensitivity, particularly for point-of-care diagnostic applications. To address these challenges, we have developed an aptamer-based electrochemical biosensing platform for the detection of SARS-CoV-2 (Omicron variant, OPV) using a steric hindrance mechanism to modulate DNA hybridization dynamics. In this work, a mixed monolayer of SARS-CoV-2-specific aptamers and single-stranded DNA (ssDNA) probes was immobilized on an electrode surface. A methylene blue-labeled ssDNA reporter was introduced to hybridize with the complementary ssDNA probe. In the absence of the target virus, hybridization occurred freely, generating a measurable electrochemical signal via square wave voltammetry (SWV). However, upon aptamer-virus binding, steric hindrance blocked the hybridization process, reducing the signal output in a concentration-dependent manner. The study was conducted by optimizing critical parameters such as reporter DNA concentration, aptamer surface density, and incubation time to maximize sensitivity and reproducibility. Viral concentrations ranging from 0 to 10⁵ copies/mL were evaluated, and the platform achieved a limit of detection (LOD) of 247 copies/mL under optimized conditions in buffer. Real-time monitoring of the electrochemical response revealed clear differences in signal kinetics across viral concentrations. At lower concentrations (up to 10³ copies/mL), the signal decreased gradually over time, while at higher concentrations (≥10⁴ copies/mL), a rapid current reduction and saturation were observed within 48 minutes. The sensor's performance was further evaluated in complex biological media, including saliva, to assess its practical applicability. Although signal attenuation was observed due to nonspecific adsorption and interference from matrix components, the platform retained its detection capability, demonstrating robust performance in saliva samples. Interestingly, slower hybridization kinetics at lower viral concentrations were attributed to competitive interactions with DNA-binding proteins in saliva, while steric hindrance effects dominated at higher concentrations. Specificity studies confirmed the assay’s high selectivity, as negligible signal responses were observed in the presence of non-specific viruses, human coronavirus 229E, human coronavirus OC43, and Influenza B. Additionally, repeatability tests indicated excellent sensor consistency, with low relative standard deviation (RSD) values (5%-10%) observed across multiple measurements. In conclusion, the steric hindrance-based electrochemical biosensing platform demonstrates excellent sensitivity and specificity, and presents an approach for leveraging aptamers in antigen-based assays. This assay has the potential to be used for rapid and point-of-care diagnostic applications.