Mercury ions (Hg²⁺) are highly toxic and pose severe risks to human health and ecosystems, necessitating sensitive detection methods for environmental monitoring. Here, we report a paper-based graphene sensor functionalized with single-stranded DNA (ssDNA) probes for Hg²⁺ detection based on T-Hg²⁺-T coordination chemistry. To elucidate the effect of probe structure on sensing performance, we designed DNA constructs with varying numbers of guanine (G) bases (3–6, designated DNA2–DNA5) in the bridging fragment and systematically evaluated their influence on hairpin stability, Hg²⁺ binding affinity, and sensor response. The DNA3-based sensor (four G bases) exhibited optimal electronic stability and sensitivity, achieving a detection limit of 0.673 pM with effective real-time monitoring capability in aqueous media. These findings highlight the critical role of DNA sequence design in T-Hg²⁺-T-based biosensors and provide a promising strategy for sensitive and selective Hg²⁺ detection in environmental samples.