Acute respiratory distress syndrome (ARDS) is a worldwide health concern. The pathophysiological features of ALI/ARDS include a pulmonary immunological response. The development of a rapid and low-cost biosensing platform for the detection of ARDS is urgently needed. In this study, we report the development of a paper-based multiplexed sensing platform to detect human NE, PR3 and MMP-2 proteases. Through monitoring the three proteases in infected mice after the intra-nasal administration of LPS, we showed that these proteases played an essential role in ALI/ARDS. The paper-based sensor utilized a colorimetric detection approach based on the cleavage of peptide-magnetic nanoparticle conjugates, which led to a change in the gold nanoparticle-modified paper sensor. The multiplexing of human NE, PR3 and MMP-2 proteases was tested and compared after 30 min, 2 h, 4 h and 24 h of LPS administration. The multiplexing platform of the three analytes led to relatively marked peptide cleavage occurring only after 30 min and 24 h. The results demonstrated that MMP-2, PR3 and human NE can provide a promising biosensing platform for ALI/ARDS in infected mice at different stages. MMP-2 was detected at all stages (30 min-24 h); however, the detection of human NE and PR3 can be useful for early- (30 min) and late-stage (24 h) detection of ALI/ARDS. Further studies are necessary to apply these potential diagnostic biosensing platforms to detect ARDS in patients.