Based on the inner filter effect mechanism of quantum dots, a ratiometric fluorescence nanoprobe was constructed for the determination of Pb(II) ion. Green emitting quantum dots conjugated with DNA substrate (DNA) acted as donors providing green fluorescence, while gold nanoparticles coupled with DNA enzyme (DNA) as acceptors quench the green fluorescence. Meanwhile, FeO nanosphere served as magnetic substrates to facilitate separation process and red fluorescence as an "inner rule" to eliminate the background signal. In the presence of Pb(II) ion, the DNA specifically recognize and capture Pb(II) ion with enhanced catalytic activity, which can cleave DNA and "turn on" the green fluorescence (I), while the red fluorescence (I) remained unchanged. In this way, the ratio of I/I reflects the Pb(II) ion in the system, enabling the quantitative and selective determination of Pb(II) ion over nine different metal ions. Under optimal conditions, the ratiometric fluorescence assay showed good linearity (R = 0.98) within the range 10 to 100 ng mL. The limit of detection (LOD) was calculated to be 1.79 pg mL (S/N = 3, n = 3, ±3.8%). The proposed fluorescence nanoprobe provides better sensitivity and accuracy than non-ratiometric signal evaluation for Pb(II) ion determination. Schematic representation of ratiometric fluorescence nanoprobe for Pb(II) ion detection using green fluorescence of I as "signal switch" and red fluorescence of I as "inner rule." Graphical abstract.