Transparent and flexible multilayer films are fabricated based on the alternating assembly of cellulose acetate (CA) and layered double hydroxide (LDH) nanoplatelets followed by thermal annealing treatment. The films exhibit tremendously enhanced oxygen barrier properties. The oxygen transmission rate (OTR) of the resulting (CA/LDH)n multilayer films can be tuned by changing the aspect ratio of high-crystalline LDH nanoplatelets from 20 to 560. The (CA/LDH)20 film displays excellent oxygen-barrier behavior with an OTR equal to or below the detection limit of commercial instrumentation (<0.005 cm3 m−2 day−1), much superior to the previously reported inorganic flake-filled barrier film. Molecular dynamics simulations reveal that a hydrogen bonding network occurs at the interface of highly oriented LDH nanoplatelets and CA molecules, accounting for the suppression of oxygen transportation and the resulting largely improved barrier behavior. In addition, the durability of (CA/LDH)n films against humidity, temperature, and light irradiation is successfully demonstrated, which would guarantee their practical application. Therefore, this work provides a facile and cost-effective strategy for the fabrication of an LDH-based oxygen barrier material, which could potentially be used in flexible displays and drug and food packaging.