Sepsis is an immune response to a microbial invasion that causes organ injury and dysfunction due to a systemic inflammatory response. Sepsis is a serious, life-threatening condition and a widely recognized global health challenge. Given its high death rate, it is critical to diagnose sepsis and start treatment as early as possible. There is an urgent need for a sensitive and rapid screening method for detecting sepsis. In this study, we investigated the use of MMP-9 as a biomarker for sepsis. A colorimetric paper-based biosensor was used for the detection of MMP-9 utilizing peptide-magnetic nanoparticle conjugates. The method is based on the cleavage of the MMP-9-specific peptide by the protease leading to the detaching of the magnetic beads from the sensor surface and changing of color. A fecal intraperitoneal (FIP) challenge was used to induce sepsis in mice, and an MMP-9 secretion was measured by taking blood and Bronchoalveolar Lavage (BAL) fluid samples at 1 h, 2 h, 4 h, and 20 h (early sepsis) post-challenge intervals. The results of the paper-based sensor for the detection of MMP-9 levels in blood samples and BAL samples were compared with ELISA and Western Blot. We found that both blood and BAL levels of MMP-9 increased immediately and could be detected as early as 1 h in FIP mice post-challenge. Our work adds evidence to the assertion that MMP-9 is a reliable biomarker for the detection of sepsis at early stages.