Metaproteomics can provide valuable insights into the functions of human gut microbiota (GM), but is challenging due to the extreme complexity and heterogeneity of GM. Data-independent acquisition (DIA) mass spectrometry (MS) has been an emerging quantitative technique in conventional proteomics, but is still at the early stage of development in the field of metaproteomics. Herein, we applied library-free DIA (directDIA)-based metaproteomics and compared the directDIA with other MS-based quantification techniques for metaproteomics on simulated microbial communities and feces samples spiked with bacteria with known ratios, demonstrating the superior performance of directDIA by a comprehensive consideration of proteome coverage in identification as well as accuracy and precision in quantification. We characterized human GM in two cohorts of clinical fecal samples of pancreatic cancer (PC) and mild cognitive impairment (MCI). About 70,000 microbial proteins were quantified in each cohort and annotated to profile the taxonomic and functional characteristics of GM in different diseases. Our work demonstrated the utility of directDIA in quantitative metaproteomics for investigating intestinal microbiota and its related disease pathogenesis.