On-line enrichment is effective for improving the sensitivity of paper-based analytical devices (PADs). Electrokinetic stacking of ionic species - anionic or cationic species, respectively, on a paper-based fluidic channel has been well demonstrated in the literature. In this work, we further demonstrated that both anionic and cationic species can be electrokinetically stacked and separated simultaneously on the same paper fluidic channel. The feasibility of the proposed method was visually demonstrated by using a colored cationic probe of Rhodamine 6G and an anionic probe of Brilliant Blue. With the introduction of a background electrolyte (BGE) consisting of weak acid and weak base salt, two electric field gradients can be developed on the same paper fluidic channel when a DC voltage was applied. Both of the anionic and cationic species from the reservoirs can be simultaneously stacked as separate bands on the two field gradients, respectively. Under optimized conditions, two orders of magnitude enrichment factors can be achieved for the anionic and cationic probes as characterized by colorimetric analysis by smartphone imaging. The applicability of this method was further demonstrated by stacking and separation of copper ions/nitrite and even amphoteric ions-proteins of phycocyanin (blue, pI 4.4)/cytochrome C (brown, pI 10.2). Potential applications can be found not only for a PAD based point of care test (POCT), but also for sample pretreatment in protein analysis considering the friendliness of the BGE to the mass spectrometer.