Deuterium with high purity can be widely used in non-radioactive isotope tracking, neutron scattering and nuclear fusion. Current approaches for deuterium separation with Pd membrane and quantum sieving-based nanoporous materials are performed at high temperature (673 K) and ultralow temperature (30 K), respectively, both requiring high energy consumption but possessing low separation efficiency. However, efficient separation of deuterium under ambient conditions is still a remaining challenge. Here, we efficiently extract high-purity deuterium from an isotopic mixture under ambient conditions via spatial confinement between Pd nanoparticles and benzene skeleton, and reducing activation energy of the reaction. In-situ FT-IR results confirm that Pd nanoparticles (ca. 1.1 nm) in crystalline porous organic framework (CPOF-1) can chemically adsorb deuterium at room temperature. Density functional theory (DFT) calculations indicated that the spatial confinement effect reduces the reaction activation energy, and leads to Pd-deuterium reaction at room temperature. This study provides a new concept for deuterium separation with low energy consumption, low cost and high efficiency, and we envision that it could be of great significance in practical applications.