Single-atom catalysts are becoming a hot research topic owing to their unique characteristics of maximum specific activity and atomic utilization. Herein, a new single-atom nanozyme (SAN) based on single Fe atoms anchored on N-doped carbons supported on carbon nanotube (CNT/FeNC) is proposed. The CNT/FeNC with robust atomic Fe-Nx moieties is synthesised, showing superior peroxidase-like activity. Furthermore, the CNT/FeNC is used as the signal element in a series of paper-based bioassays for ultrasensitive detection of H2 O2 , glucose, and ascorbic acid. The SAN provides a new type of signal element for developing various biosensing techniques.