The sensitive detection of Pb2+ is of significant importance for food safety, environmental monitoring, and human health care. To this end, a novel fluorescent biosensor, DNAzyme-functionalized R-phycoerythrin (DNAzyme-R-PE), was presented for Pb2+ analysis. The biosensor was prepared via the immobilization of Iowa Black® FQ-modified DNAzyme–substrate complex onto the surface of SPDP-functionalized R-PE. The biosensor produced a minimal fluorescence signal in the absence of Pb2+. However, Pb2+ recognition can induce the cleavage of substrate, resulting in a fluorescence restoration of R-PE. The fluorescence changes were used to measure sensitively Pb2+ and the limit of detection was 0.16 nM with a linear range from 0.5–75 nM. Furthermore, the proposed biosensor showed excellent selectivity towards Pb2+ even in the presence of other metal ions interferences and was demonstrated to successfully determine Pb2+ in spiked lake water samples.