Hydrogen peroxide (H2O2) photoproduction in seawater with metal-free photocatalysts derived from biomass materials is a green, sustainable, and ultra environmentally friendly way. However, most photocatalysts are always corroded or poisoned in seawater, resulting in a significantly reduced catalytic performance. Here, we report the metal-free photocatalysts (RUT-1 to RUT-5) with in-situ generated carbon dots (CDs) from biomass materials (Rutin) by a simple microwave-assisted pyrolysis method. Under visible light (λ ≥ 420 nm, 81.6 mW/cm2), the optimized catalyst of RUT-4 is stable and can achieve a high H2O2 yield of 330.36 µmol/L in seawater, 1.78 times higher than that in normal water. New transient potential scanning (TPS) tests are developed and operated to in-situ study the H2O2 photoproduction of RUT-4 under operation condition. RUT-4 has strong oxygen (O2) absorption capacity, and the O2 reduction rate in seawater is higher than that in water. Metal cations in seawater further promote the photo-charge separation and facilitate the photo-reduction reaction. For RUT-4, the conduction band level under operating conditions only satisfies the requirement of O2 reduction but not for hydrogen (H2) evolution. This work provides new insights for the in-situ study of photocatalyst under operation condition, and gives a green and sustainable path for the H2O2 photoproduction with metal-free catalysts in seawater.