Carbon nanozymes are catalytic carbon nanomaterials with intrinsic enzyme-like activities. They are advantageous over their natural counterparts in terms of higher stability, lower preparation cost, and better robustness. However, the peroxidase-like activities of the most developed carbon nanozymes were moderate due to the imperfection of active centers and limited tuning strategies. Herein, we designed a novel class of efficient peroxidase-mimicking carbon nanozymes with nitrogen atom doping. The N-doped carbon nanozymes were facilely synthesized by direct pyrolysis of different nucleobases at controlled temperatures without other treatments. A high ratio of nitrogen atoms was doped into the carbon skeleton. For example, 8.77 wt% of N remained in the guanine-derived carbon nanozyme with a pyrolysis temperature of 900 °C. The dominant graphitic N species greatly boosted the peroxidase-like activities of nucleobase-derived carbon nanozymes. Moreover, nucleobases are cheap, abundant, and environmentally friendly. We have demonstrated that nitrogen-rich nucleobases are ideal starting materials for the large-scale and cost-effective synthesis of N-doped carbon nanozymes. The carefully designed N-doped carbon nanozymes with superior activities were further used to construct effective biosensors for bioactive molecules (i.e., H2O2 and glucose). Highly sensitive and selective detection of H2O2 and glucose was achieved using the N-doped carbon nanozymes as efficient peroxidase mimics. This study offers an economical and sustainable approach for the scalable preparation of N-doped carbon nanozymes and creates a new path for the rational design of efficient peroxidase-mimicking carbon nanozymes by heteroatom doping.