Abstract: To address the need for marine biofouling prevention, this study developed a novel environmentally friendly, long-term-release cuprous oxide-cellulose nanofiber (Cu₂O-CNF) composite antifouling agent. Using the ascorbic acid reduction method, this work investigated the effects of surfactant type and copper source type on the particle size and morphology of Cu₂O particles. Results indicate that adding polyvinylpyrrolidone (PVP) and using copper sulfate pentahydrate enabled the preparation of uniform nano-Cu₂O particles with an average size of (43.3±8.5) nm. To suppress nano-Cu₂O agglomeration and enhance applicability, a series of Cu₂O-CNF composite antifouling agents were prepared through in-situ synthesis, loading Cu₂O onto carboxylated cellulose nanofibers (CNF). When the cellulose nanofiber (CNF) loading was moderate (Cu₂O-0.5CNF, mass ratio 10:1), the resulting composite particles exhibited improved uniformity and a markedly reduced average size of (38.2±7.7) nm. Antibacterial performance tests demonstrated that both the prepared nano-Cu₂O and Cu₂O-0.5CNF composite exhibited over 99% inhibition rates against Escherichia coli and Staphylococcus aureus. Utilizing the electrostatic interaction between carboxyl groups (—COOH) on the CNF surface and Cu²⁺ ions, the composite antifoulant achieved a significantly lower initial Cu²⁺ release amount (0.67 mg/L) compared to pure Cu₂O (1.86 mg/L), effectively regulating the ion release kinetics. This study provides an effective strategy for developing high-performance and sustainable composite antifouling materials based on nano-Cu₂O and biomass carriers.