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Abstract. 3D printing can significantly improve the current fabrication techniques for microfluidic devices due to its ability to create truly 3D structures in a single step. In this study, an active pneumatic microfluidic mixer was designed and fabricated using an extrusion-based 3D printer for rapid detection of Listeria monocytogenes. The printed material of the mixer is flexible, semi-transparent and inexpensive. The fabrication time is significantly shorter than the traditional micromolding process. The printed mixer consists of two pneumatic air chambers and one mixing chamber designed for a fluidic sample size of 100 μL. The length, width, and height of the mixer chip are 13, 12.7, and 9 mm, respectively. The performance of the mixer was tested for different actuation frequencies and pneumatic pressures. The completed 3D-printed mixer was successfully applied to the colorimetric detection of L. monocytogenes for a concentration range from 10² to 10⁸ cfu/mL using an enzyme-linked immunosorbent assay. The experimental results showed the microfluidic mixer could enhance the mixing efficiency of the fluidic sample through pneumatically actuated diaphragms. In addition, the mixer could accelerate the color development caused by target L. monocytogenes, and the observed color changes could be discriminated within 5 minutes by naked eye. The present work will contribute to the development and optimization of a prototype for rapid detection of L. monocytogenes in food samples. It provides an effective technical approach to realize the fabrication of low-cost microfluidic chips for efficient reagent mixing in microscale biochemical detection systems.