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Nanoporous gold film (NPGF) has shown great potential for photonics, catalysis, sensing, and renewable energy. Herein, we report a monolayered NPGF with tunable electrocatalytic activity for improved methanol electrooxidation and H2O2 detection. The NPGFs were fabricated via interfacial self-assembly of Au nanoparticles (NPs) with different sizes and a subsequent light welding process. Amongst the NPGFs made from 16, 30, 55, and 70 nm Au NPs (denoted as x nm-NPGF, where x represents the size for the Au NPs), the 16 nm-NPGF shows the highest electrocatalytic activity, which is attributed to the synergistic contribution from its largest surface area, high conductivity, and the smallest hyperboloid-like ligaments that expose more high-index facets. Consequently, the optimized 16 nm-NPGF delivers a large current density of 151 μA cm−2 (at 0.22 V vs. saturated calomel electrode in 0.1 M KOH and 1.0 M methanol) for methanol electrooxidation. Moreover, it as well shows a good linear range (from 0.005 to 45 mM) for H2O2 detection, reaching a detection limit of 2.56 μM and sensitivity of 156.6 μA mM−1 cm−2. Our studies, thus, offer a low-temperature, solution-processable approach to fabricate monolayered NPGF from easily available Au NPs, without harsh dealloying process.