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This study presents a rapid microfluidic paper-based analytical device (μPAD) capable of simultaneously monitoring Gram-negative bacteria and nitrite ions (NO₂^–) for water quality monitoring. We utilize gold nanoparticles (AuNPs) functionalized with polymyxin molecules (AuNPs@polymyxin) to cause color change due to aggregation for the detection of Gram-negative bacteria, and antiaggregation in the presence of o-phenylenediamine (OPD) for NO₂^– detection. In this study, Escherichia coli (E. coli) serves as the model of a Gram-negative bacterium. Using the developed μPADs, the color changes resulting from aggregation and antiaggregation reactions are measured using a smartphone application. The linear detection ranges from 5.0 × 10² to 5.0 × 10⁵ CFU/mL (R² = 0.9961) for E. coli and 0.20 to 2.0 μmol/L (R² = 0.995) for NO₂^–. The detection limits were determined as 2.0 × 10² CFU/mL for E. coli and 0 …