Abstract: The coexistence of multiple heavy metal ions poses a serious threat to human health and environmental safety, therefore it is particularly important to establish sensitive, rapid, and reliable analysis methods. Combining the target recognition elements including aptamer and DNAzyme with Y-shaped DNA structure and magnetic separation, a fluorescent biosensor was constructed for simultaneous detection of Pb²⁺ and Cd²⁺. The Pb²⁺ DNAzyme strand labeled with Cy5 was hybridized to the Cd²⁺ aptamer strand modified with FAM to form a stable Y-shaped DNA structure. This structure was anchored to the surface of magnetic beads by the strong binding force between biotin and streptavidin. In the absence of Pb²⁺ and Cd²⁺, the fluorescent groups of Cy5 and FAM remained stable in the Y-shaped DNA structure and were separated from the solution together with MBs by magnetic separation, hence, no significant fluorescence signal can be detected in the supernatant. When Pb²⁺ and Cd²⁺ were introduced, DNAzyme specifically recognized Pb²⁺ and the S-DNA was cleaved to release the ssDNA labeled with Cy5. Meanwhile, Cd²⁺ is competitively bound to the aptamer to separate it from the Y-shaped DNA structure. After magnetic separation, the fluorescence signals of Cy5 and FAM could be detected in the supernatant. Based on the above strategy, a novel fluorescence sensor with simple steps, high sensitivity, and good stability has been successfully developed. Under the optimal conditions, the prepared fluorescence biosensors showed a linear response to Pb²⁺ and Cd²⁺ in the range of 0.5 to 100 μmol/L and 0.01 to 100 μmol/L, respectively. The limit of detection (LOD) of Pb²⁺ and Cd²⁺ was 9.7 nmol/L and 0.4 nmol/L, respectively. In addition, the RSD was in the range of 3.3% to 5.4% and the recoveries ranged from 82.33% to 107.25% in actual samples. Therefore, the fabricated fluorescent biosensors are capable to provide a powerful tool for the multiplex detection of heavy metal ions.