Molybdenum disulfide (MoS₂) with single- and odd-numbered layers is a novel piezocatalyst, and its piezocatalytic molecular oxygen activation, which produces reactive oxygen species (ROS), has been considered a promising and low-cost strategy for environmental remediation. However, its performance is still far from satisfactory, and the limited knowledge regarding its molecular oxygen activation process significantly impedes its further development. Herein, several numbered layers of Co-doped MoS₂ ultrathin nanosheets (UNs) with a thickness of 3.2 nm were successfully fabricated via hydrothermal synthesis. The single Co atom-doped odd-numbered layers of MoS₂ nanosheets strongly interacted with adsorbed oxygen molecules for a highly efficient generation of ROS in the piezocatalytic degradation process. The dopant-induced enhanced carrier density (electron density) of Co-doped MoS₂ was 7.7 × 10¹⁸ cm^–3, compared with 2.9 × 10¹⁶ cm^–3 for bare MoS₂. Moreover, the interfacial action of Co-doped MoS₂ nanosheets on directional molecular oxygen activation properties were predicted by DFT calculation and monitored by generated reactive oxygen species (ROS) evolution. Co-doped MoS₂ decomposed tetracycline (an antibiotic) by 99.8% in 15 min through shaking vibration in the dark. It was found that Co active sites can facilitate the one-electron reduction of molecular oxygen activation by introducing a Co²⁺/Co³⁺ redox couple. In addition, a tentative mechanism was proposed to explain the origin of the piezocatalytic enhancement in Co-doped MoS₂. Thus, in order to meet the requirements in the field of wastewater pollutant remediation, the current research effort may provide guidelines for constructing 2D TMDCs piezoelectric catalysts and comprehending the mechanisms of the directional piezoelectric catalytic generation of reactive oxygen species through the doping route.