Preferential oxidation of CO (CO-PROX) is an efficient method to eliminate residual CO in the feed stream to avoid Pt poisoning in proton-exchange-membrane fuel cells (PEMFCs), in which the development of high-performance, low-cost catalysts remains a big challenge. Herein, we report highly active spinel-like MnCoOx catalysts derived from layered double hydroxide (LDH) precursors, which are featured with abundant octahedron-distorted lattice oxygen. Impressively, the optimal catalyst MnCoOx-300 achieves the selective and complete removal of CO from a H2-rich stream at 80 °C, within a wide operation temperature window (80–200 °C, matching well with PEMFCs) at a rather high space velocity (80,000 h–1). This performance, to the best of our knowledge, outperforms previously reported non-noble metal catalysts and even exceeds the state-of-the-art CuO/CeO2 system in the CO-PROX technology. A comprehensive investigation based on in situ Raman, in situ XAFS, in situ TPD-Mass, and in situ DRIFTS reveals that the Cooct3+–O2––Mnoct4+ structure in MnCoOx-300 serves as the intrinsic active site that facilitates preferential oxidation: the lattice oxygen participates in the oxidation of CO to produce CO2 and oxygen vacancy (Ov), followed by the replenishment of oxygen species from aerial oxygen (the rate-determining step) to regenerate Cooct3+–O2––Mnoct4+. Isotopic 18O kinetic studies and in situ DRIFTS substantiate that the reaction temperature plays a crucial role in the competitive oxidation of CO vs H2 at the same active site. This work provides a successful paradigm for the design and preparation of transition metal oxide catalysts toward the CO-PROX reaction, which shows potential applications in hydrogen purification for PEMFCs.