Aim. Tissue kallikrein (TK) protein content in plasma has been shown to be negatively associated with both incident and recurrent strokes. The aims of this study were to develop a novel method for detecting TK activity and to investigate its association with event-free survival over 5 years in Chinese first-ever stroke patients. Methods. We designed a case-control study with 321 stroke patients (174: ischemic stroke, 147: hemorrhagic stroke) and 323 healthy local controls. TK activity was measured by a novel assay utilizing the immunological characteristics of TK and the catalysis of benzoyl arginine ethyl ester hydrochloride (BAEE). Results. TK protein levels above 0.200 mg/L in plasma were not associated with urinary TK activity or the risk of stroke recurrence. TK activity was significantly lower in stroke patients compared with controls (1.583 +/- 0.673 Eu/mL versus 1.934 +/- 0.284 Eu/mL, P < 0.001). After adjusting for traditional risk factors, TK activity was negatively associated, in a dose-responsemanner, with the risk of overall stroke recurrence and positively associated with event-free survival during a 5-year follow-up (relative risk (RR), 0.69; 95% CI, 0.57-0.84; P < 0.001). Conclusions. Our findings suggest that urinary TK activity may be a stronger predictor of stroke recurrence than plasma TK levels.