Activation of molecular oxygen plays a crucial role in natural organisms and the modern chemical industry. Herein, we report a Mn–Co dual-single-atom catalyst that exerts a specific synergy in boosting O₂ activation by collaboration between two distinct types of activation sites. Taking the oxidative esterification of the biomass platform 5-hydroxymethylfurfural (HMF) as the model reaction, the activation of O₂ is demonstrated through transforming O₂ into a reactive superoxide anion radical (O₂^•–) on Co–N₄ sites and, meanwhile, by reversible consumption and supplement of coordinated surface oxygen as a new type of reactive oxygen species (ROS) on N,O-coordinated single-atom Mn sites (Mn–N_xO_y). EXAFS analysis results show a longer average Mn–O bond distance at near 2.19 Å, which makes the breaking and formation of surface Mn–O bonds easier to cycle. Control experiments support that such Mn–O bonding conditions could facilitate H-elimination of C–H in HMF. The co-existence of two types of ROS effectively matches the oxidation of hydroxyl and aldehyde groups, and thus, the overall reaction is boosted in excellent yield of diester (95.8%) with an extremely high carbon balance. This study represents a rare example of taking advantage of the synergy of the diatomic catalyst for activating O₂ by two types of activation pathways.