We describe here a portable DNAzyme-based optical fiber evanescent wave biosensor (OFEWB) for the rapid and sensitive detection of Pb 2+ in human serum. Unlike other biosensors, the OFEWB dispensed with the complicated process of attaching biometric elements to the optical fiber, and the optical fiber directly acted as a transducer to transmit the excitation light and simultaneously collected the fluorescence, which could simplify the detection process, avoid the susceptibility to interference from complex environments and strengthen the reusability of the biosensor. The fluorescence (Cy3) labelled substrate sequence (GR-5S-Cy3) could be cleaved under the catalysis of the GR-5 DNAzyme sequence (GR-5E-BHQ2) in the presence of Pb 2+ ; then the released fluorescence labelled fragments could be directly excited and detected by the OFEWB due to the high transmission efficiency of the excitation light and fluorescence in the OFEWB. Several key factors affecting Pb 2+ detection were investigated in detail and optimized. Under the optimal conditions, the LOD of Pb 2+ in human serum was 9.34 nM (equivalent to 93.4 nM in whole serum) with a detection range of 0-120 nM. The possible matrix interference was evaluated with different spiked human serum samples, and the recovery of Pb 2+ ranged from 74.4% to 112.5% with RSD < 14.8%, implying this method had excellent practicability and could be potentially used in analyzing some biomedical samples.