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This paper proposes a new “twisted” 3D microfluidic mixer fabricated by a laser writing/microfabrication technique. Effective and efficient mixing using the twisted micromixers can be obtained by combining two general chaotic mixing mechanisms: splitting/recombining and chaotic advection. The lamination of mixer units provides the splitting and recombination mechanism when the quadrant of circles is arranged in a two-layered serial arrangement of mixing units. The overall 3D path of the microchannel introduces the advection. An experimental investigation using chemical solutions revealed that these novel 3D passive microfluidic mixers were stable and could be operated at a wide range of flow rates. This micromixer finds application in the manipulation of tiny volumes of liquids that are crucial in diagnostics. The mixing performance was evaluated by dye visualization, and using a pH test that determined the chemical reaction of the solutions. A comparison of the tornado-mixer with this twisted micromixer was made to evaluate the efficiency of mixing. The efficiency of mixing was calculated within the channel by acquiring intensities using ImageJ software. Results suggested that efficient mixing can be obtained when more than 3 units were consecutively placed. The geometry of the device, which has a length of 30 mm, enables the device to be integrated with micro total analysis systems and other lab-on-chip devices.