The surgical connection of blood vessels, anastomosis, is a critical procedure in many reparative, transplantation, and reconstructive surgical procedures. However, effective restoration of circulation is complicated by pathological clotting (thrombosis) or progressive occlusion due to excess cell proliferation that often leads to additional surgeries and increases morbidity and mortality risk for patients. Pharmaceutical agents have been tested to prevent these complications, but many have unacceptable systemic side effects. Therefore, an alternative approach to deliver these drugs at the site of injury in a controlled manner is necessary. The objective of this study was to develop electrospun nanofibers composed of polyester poly(lactide- co-glycolide) (PLGA), poly(ethylene oxide) (PEO), and positively charged copolymer, poly(lactide- co-glycolide)- graft-polyethylenimine (PgP) for electrostatic binding and release of heparin for application as an antithrombotic microvascular suture. PgP was synthesized with different coupling ratios between PLGA and branched polyethylenimine (bPEI) to obtain PgP (∼1 PLGA grafted to 1 bPEI) and PgP (∼3.7 PLGA grafted to 1 bPEI). Nanofiber yarns (PLGA/PEO/PgP and PLGA/PEO/PgP) were fabricated by electrospinning. Heparin immobilization on the positively charged nanofiber yarns was visualized using fluorescein-conjugated heparin (F-Hep), and the amount of immobilized F-Hep was higher on both PLGA/PEO/PgP and PLGA/PEO/PgP than yarns without PgP (PLGA/PEO). We also found that F-Hep was released from both PgP-containing yarns in a sustained manner over 20 days, while over 60% of F-Hep was released within 4 h from PLGA/PEO. Finally, we observed that heparin-eluting nanofiber yarns with both PgP and PgP showed significantly longer clotting times than nanofiber yarns without PgP. The clotting time of PLGA/PEO/PgP was not significantly different than that of free heparin (0.5 μg/mL). These results show that heparin-eluting electrospun nanofiber yarns may offer a basis for the development of microvascular sutures with anticoagulant activity.