Biomaterials that come in contact with blood require excellent antithrombogenicity. The antithrombogenicity of newly developed biomaterials is evaluated by in vitro tests and animal experiments. However, no in vitroevaluation system has been established that can sufficiently evaluate the complicated mechanisms of thrombus formation within the body. Even in animal experiments, it is difficult to observe the chronology of thrombus formation in real time. In this study, we aimed to develop a method for the evaluation of antithrombogenicity of biomaterials. To this end, we examined the optical detection of platelet aggregation occurring on the surface of biomaterials. An apparatus that optically detects platelet aggregation in a test tube or in an in vitro blood circulation circuit was constructed. We used the luciferin-luciferase (L-L) reaction that emits light with an intensity proportional to the concentration of adenosine triphosphate (ATP) s released from platelets after aggregation on the surface of biomaterials. Blood with L-L luminescent reagent added was allowed to aggregate, and chronological changes in intensity of the resulting emission were detected using a photomultiplier tube. This experimental apparatus was able to detect the emission intensity corresponding to the amount of platelet aggregation. The results of in vitro blood circulation tests using a polyvinyl chloride tube or a porcine carotid artery showed a significant and strong correlation between maximum emission intensity based on platelet aggregation and dry weight of thrombus formed within the circulation circuit (r = 0.727, p = 0.007). Thus, this experimental system was proven to be useful as an alternative to animal experiments for the evaluation of antithrombogenicity of biomaterials.