To improve the efficiency of mixing under laminar flow with a low Reynolds number (Re), a novel three-dimensional Horseshoe Transformation (3D HT) was proposed as the basis for the design of a micromixer. Compared with the classical HT, the Lyapunov exponent of the 3D HT, which was calculated based on a symbolic dynamic system, proved the chaotic enhancement. Based on the 3D HT, a micromixer with a mixing length of 12 mm containing six mixing units was obtained by sequentially applying "squeeze", "stretch", "twice fold", "inverse transformation", and "intersection" operations. Numerical simulation and Peclet Number (Pe) calculations indicated that when the squeeze amplitude 0 < α < 1/2, 0 < β < 1/2, the stretch amplitude γ > 4, and Re ≥ 1, the mass transfer in the mixer was dominated by convective diffusion induced by chaotic flow. When Re = 10, at the outlet of the mixing chamber, the simulated mixing index was 96.4%, which was far less than the value at Re = 0.1 (σ = 0.041). Microscope images of the mixing chamber and the curve trend of pH buffer solutions obtained from a mixing experiment were both consistent with the results of the simulation. When Re = 10, the average mixing index of the pH buffer solutions was 91.75%, which proved the excellent mixing efficiency of the mixer based on the 3D HT.