Thrombolytic therapy is limited by rapid enzyme deactivation, off-target bleeding, and inefficient thrombus targeting. Here, we report a DNA-programmed gold nanoparticle (AuNP) platform that enables precise spatial control of tissue plasminogen activator orientation and thrombin-responsive regulation for synergistic thrombolysis and anticoagulation. By fine-tuning the polyA chain length and hybridization density, we identified the poly20A configuration as optimal for maximizing tPA (tissue plasminogen activator) loading and enzymatic accessibility. Embedded tPA exhibited superior stability and activity retention compared with exposed tPA, maintaining a 1.4-fold higher catalytic efficiency and enhanced resistance to PAI-1 inhibition. The thrombin-responsive DNA interface enabled targeted activation at thrombus sites, achieving a dose-dependent thrombolytic response. In vitro and in vivo studies demonstrated potent thrombus dissolution and thrombin inhibition, leading to a 75% reduction in thrombus area, 80% survival rate, and minimal hemorrhagic risk in mouse pulmonary embolism and femoral vein thrombosis models. This dual-function system, combining site-specific thrombin binding and protected tPA delivery, establishes a programmable and modular nanoplatform for precision thrombolysis and anticoagulation, offering a generalizable strategy for targeted vascular nanotherapeutics.