Electrochemical biosensing combined with screen-printed paper technology provides an effective strategy for the quantitative analysis of metal ions, particularly suitable for in-situ determination. In this study, a disposable, economical and user-friendly screen-printed dual-working electrodes (SPDEs) aptasensor system for the determination of lead ions (Pb2+) and mercury ions (Hg2+) was described. The sensor system consists of dual-working electrodes, a counter electrode and a reference electrode fabricated using screen printing technology. The aptasensor demonstrates high sensitivity and selectivity towards Pb2+ and Hg2+, owing to the larger specific surface area (1334.7 m2/g) and excellent conductivity of mesoporous carbon nanoparticles (MCNs). Additionally, the specific recognition ability and high affinity of G-quadruplex and T-Hg2+-T structures for Pb2+ and Hg2+, respectively, further enhance the performance. Under optimized experimental conditions, the aptasensor achieves a low limit of detection (LOD) of 0.088 ng/mL for Pb2+ and 0.0007 ng/mL for Hg2+ (S/N = 3), a wide linear range (0.1–1000.0 ng/mL for Pb2+ and 0.001–100.0 ng/mL for Hg2+), and high accuracy (relative standard deviation, RSD≤10.0 %) and selectivity. Importantly, the SPDEs aptasensor can detect Pb2+ and Hg2+ in a sample without competition and interference, achieving accuracy comparable to that of commercial inductively coupled plasma mass spectrometry (ICP-MS). These advantages make the SPDEs aptasensor highly suitable for practical applications in environmental monitoring, food safety, and clinical diagnosis.