A simple double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) was designed for the simultaneous determination of six metal ions-Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II)-for the first time. The 3D μPAD was composed of two paper layers: a top pretreatment layer and a bottom colorimetric detection layer. The sample solution added to the central sample reservoir of the 3D μPAD could be automatically divided into eight flow pathways and be automatically pretreated while flowing through the pretreatment zones located in the microfluidic channels, and automatically carried out the chromogenic reactions after reaching the detection zones. Random diffusion of the chromogenic reagents was effectively prevented by transport of the pretreated sample solution to the detection zones through 3D microfluidic channels with an L-type circuitous flow route design, resulting in highly increased color uniformity and reproducibility. Combined with use of a flat LED lamp as an upward lighting source and a smartphone as a convenient detector, improved color perception, highly enhanced sensitivity, and an extended detection range were obtained. Finally, the double-layered 3D μPAD was applied to the multiplexed determination of the six metal ions in mixtures and environmental samples with satisfactory results. Detection limits as low as 0.2, 0.3, 0.1, 0.03, 0.08, and 0.04 mg/L for Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II) detection, respectively, were achieved, which are about one order of magnitude lower than obtained with previously reported μPADs for the detection of metal ions. The present 3D μPAD is simple, fast, selective, sensitive, and user-friendly, and holds great application potential for multiplexed on-site analysis.