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This protocol provides insights into the rapid, low-cost, and largescale fabrication of polymer microfluidic chips containing three-dimensional microstructures used in point-of-care devices for applications such as detection of pathogens via molecular diagnostic methods. The details of the fabrication methods are described in this paper. This study offers suggestions for researchers and experimentalists, both at university laboratories and in industrial companies, to prevent doom fabrication issues. For a demonstration of bio-application in point-of-care testing, the 3D microarrays fabricated are then employed in multiplexed detection of <i>Salmonella</i> (<i>Salmonella</i> Typhimurium and <i>Salmonella</i> Enteritidis), based on a molecular detection technique called solid-phase polymerase chain reaction (SP-PCR).