In this study, we present a novel Full Window DIA (FW-DIA) method designed to address the challenges associated with data-independent acquisition in top-down proteomics. The FW-DIA approach significantly enhances fragment signal intensities by approximately 50-fold and increases protein sequence coverage threefold. The efficacy of FW-DIA was demonstrated through the analysis of a five-protein mixture under native conditions, as well as ribosomal proteins from Escherichia coli (E. coli).