In this work, a novel dual photoelectrochemical/colorimetric cyto-analysis format was first introduced into a microfluidic paper-based analytical device (μ-PAD) for synchronous sensitive and visual detection of H2O2 released from tumor cells based on an in situ hydroxyl radicals ((•)OH) cleaving DNA approach. The resulted μ-PAD offered an excellent platform for high-performance biosensing applications, which was constructed by a layer-by-layer modification of concanavalin A, graphene quantum dots (GQDs) labeled flower-like Au@Pd alloy nanoparticles (NPs) probe, and tumor cells on the surface of the vertically aligned bamboo like ZnO, which grows on a pyknotic Pt NPs modified paper working electrode (ZnO/Pt-PWE). It was the effective matching of energy levels between GQDs and ZnO levels that lead to the enhancement of the photocurrent response compared with the bare ZnO/Pt-PWE. After releasing H2O2, the DNA strand was cleaved by (•)OH generated under the synergistic catalysis of GQDs and Au@Pd alloy NPs and thus, reduced the photocurrent, resulting in a high sensitivity to H2O2 in aqueous solutions with a detection limit of 0.05 nmol observed, much lower than that in the previously reported method. The disengaged probe can result in catalytic chromogenic reaction of substrates, resulting in real-time imaging of H2O2 biological processes. Therefore, this work provided a truly low-cost, simple, and disposable μ-PAD for precise and visual detection of cellular H2O2, which had potential utility to cellular biology and pathophysiology.