Rapid and sensitive methods for the detection of pathogenic microorganisms are required to monitor contamination in milk and assure public health. A colorimetric antibody-based stack pad system (biosensor) was developed to detect microbiological content of milk based on nitrocellulose membranes. This modification of the lateral flow immunoassay uses a multiple-membrane biosensor containing (from top to bottom): a sample pad, a conjugation pad (with anti-Escherichia coli antibodies conjugated to horseradish peroxidase), six Escherichia coli strain DH5α bacteria-blocking layers and an absorption pad with dry substrate. Different milk samples were tested, with and without DH5α bacteria. This multistep diagnostic assay was up to 1000-fold more sensitive than the standard laboratory ELISA. This is the first step in the development of a simple and portable device for milk-safety monitoring during all processing stages, with the aim of ensuring fresh and healthy milk for consumption.