Paper-based analytical device (PAD) with image-based colorimetric detection is advantageous in low-cost, fast, simple and suitable for on-site rapid analysis. However, the detection capability is normally limited. In this paper, a new method was proposed that combined a complex reaction and electrokinetic stacking, and the sensitivity of PAD was effectively increased as demonstrated by the colorimetric detection of Cu2+ with smartphone. A T-type paper fluidic channel was designed to realize the on-line complex reaction between polyethyleneimine (PEI) and Cu2+. Under electric field, positively charged PEI and Cu2+ were driven through the two channels and eventually met on the same paper fluidic channel to form the positively charged complex of dark blue color. Meanwhile, the complex was stacked into a narrow and enhanced color band by field amplification effect. Under optimal experiment conditions, the band intensity was increased by three orders of magnitude compared with that of direct Cu2+ imaging on the same paper substrate. A linear relationship in the range of 0.1–1.0 mM Cu2+ was obtained (R2 = 0.991), and a limit of detection of 30 μM was achieved, which is close to that by a desktop spectrophotometer. The selectivity of this method to other co-existing metal ions was also investigated. This work shows that by integration of a complex reaction and electro kinetic stacking, the sensitivity of image-based colorimetric detection can be greatly enhanced for rapid detection of Cu2+ by PAD.