Polylysine has broad biomedical applications, though little is known about its hemocompatibility. Here, we studied the influence of polylysine on human red blood cells (RBCs) and blood clotting. We observed the morphology and aggregation and determined the hemolysis of RBCs incubated with polylysine. Plasma coagulation in the presence of polylysine was evaluated by measuring the activated partial thromboplastin time (APTT) and prothrombin time (PT). Human whole blood coagulation in the presence of polylysine was evaluated with the thromboelastograph (TEG). We found that polylysine at 0.01 mg/mL did not result in RBC aggregation or morphological change, while polylysine at ≥ 0.1 mg/mL caused RBC aggregation. The RBCs did not lyze in the presence of 0.01–0.5 mg/mL of polylysine. Polylysine at 0.001 mg/mL did not cause a significantly different APTT from the control, while polylysine at ≥ 0.01 mg/mL caused a significantly higher APTT than the control. Polylysine at ≤ 0.1 mg/mL did not cause a significantly different PT from the control, while polylysine at 1 mg/mL caused a significantly higher PT than the control. TEG parameters for whole blood coagulation in the presence of 0.01 mg/mL polylysine were within the normal range; while polylysine ≥ 0.1 mg/mL caused one or more abnormal TEG parameters. From these results, the effect of polylysine on RBC aggregation and blood coagulation was concentration-dependent. The results provide important information for the biomedical applications of polylysine.