The rapid advancement of air/water interface coatings transcends multiple domains. These coatings, valued for expansive coverage, facile acquisition, and economic efficiency, hold pivotal roles in nanosurface engineering. Conventional approaches to achieving 2D periodic structures, such as electron-beam lithography, suffer from cost and complexity challenges. Nanosphere lithography presents an alternative avenue by harnessing the inherent self-assembly propensity of colloidal nanoparticles and offers simplicity, cost-effectiveness, and compatibility with various techniques. The use of nanosphere lithography in coating metal templates gains traction in plasmonics because of its simplicity and speed. The fusion of plasmonics and biosensing enables label-free detection with unprecedented sensitivity, revolutionizing areas such as medicine, diagnostics, and environmental monitoring. In the realm of label-free biosensing, attaining uniform large-area fabrication is critical for robust sensing signals. Detecting spectral variations in low analyte concentrations necessitates a precise response profile. The creation of a potent and accessible local electromagnetic field is vital for enhancing light−biomolecule interactions. We introduce a hybrid plasmonic substrate via nanosphere lithography, combining antenna and aperture responses. The hybrid plasmonic design caters to the prerequisites of efficient label-free biosensing, uniform large-scale fabrication, narrow spectral response, and strong local electromagnetic fields. The hybrid plasmonic substrate promises to advance label-free biosensing capabilities by integrating antenna and aperture responses, facilitating sensitive and robust biosensing applications. Demonstrating these parameters resulted in a high refractive index sensitivity of up to 553 nm/RIU. The hybrid nature further facilitated an impressive detection limit of 0.5 ng/mL for label-free protein IgG detection.