An ultra-efficient biocatalytic peroxidase-like Au-based single-atom nanozyme (Au-SAzymes) has been synthesized from isolated Au atoms on black nitrogen doped carbon (Au-N-C) using a simple complexation-adsorption-pyrolysis method. The atomic structure of AuN centers in black carbon was revealed by combined high-resolution transmission electron microscopy/high-angle annular dark-field scanning transmission electron microscopy. The Au-SAzymes showed a remarkable peroxidase activity with 1.7 nM as Michaelis-Menten constant, higher than most previously reported SAzyme activity. Density functional theory and Monte Carlo calculations revealed the adsorption of HO on AuN with formation of OH* and O*. Molecular recognition was greatly enhanced via label-free integration of thiol-terminal aptamers on the surface of single Au atoms (Aptamer/Au-SAzyme) to design off-on ultrasensitive aptananozyme-based sensor for detecting thrombin and CRP with 550 pM and 500 pg mL limits of detection, respectively. The Aptamer/Au-SAzyme showed satisfactory accuracy and precision when applied to the serum and plasma of COVID-19 patients. Due to the maximum Au atom utilization, approximately 3636 samples can be run per 1 mg of gold, highlighting the commercialization potential of the developed Aptamer/Au-SAzyme approach.