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There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L₁₆ (5⁴) Taguchi design to obtain results in hours without PCR amplification. To determine the level of <i>Escherichia coli</i> (<i>E. coli</i>) contamination, after induction with 150 &#181;L of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl &#946;-<span style="font-variant: small-caps;">d</span>-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by &#946;-galactosidase from <i>E. coli</i> and dimerized in the solution to yield a blue color after incubation at 40 &#176;C. Color intensity was correlated with the level of <i>E. coli</i> contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 10² colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the $3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 10² CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 10⁴ CFU/mL, with a percentage of false positives to be determined in future efforts.