To achieve rapid and uniform mixing of trace samples, based on the mathematical model of “Horseshoe Transformation”, a series of operations on fluids, such as “extrusion stretching”, “curved folding”, and “inverse transformation-intersection” were carried out.Theoretical calculation show that when squeeze amplitude 0<λ <1/2 and stretch amplitude μ>2, the transformation could induce chaos flow successfully under laminar flow with low Reynolds number. Based on theoretical calculations, a mixer which consists of 4 mixing units with the effective mixing length of 13 mm was designed and manufactured. The numerical simulation results by COMSOL show that when Re≥1 and the Peclet Number Pe≥10, the chaotic flow intensity increased with the flow rate and gradually became the main factor to promote mixing. When Re=10, after passing through 4 “Horseshoe Transformation” mixing units, the concentration variance σ=0.054, and the mixing effect was nearly uniform. The visualization test of mixer chip show that the color change of the tracer photographed by the fluorescence microscope was consistent with the surface concentration cloud in the simulation result. Moreover, the mixing test of different pH value samples proved that the micromixer based on the “Horseshoe Transformation” can generate chaos flow and achieve a satisfactory mixing effect.