Blood flow properties play important roles in the regulation and formation of thrombus. To evaluate the influence of blood flow on thrombus formation in haemophilia, whole blood samples were obtained from FVIII-deficient (FVIII−/−) and wild-type (FVIII+/+) mice (n = 6 respectively), and from six human volunteers. Anti-FIXa aptamer was added to human blood to model acquired haemophilia B. Recalcified whole blood samples containing corn trypsin inhibitor and danaproid were perfused over the microchip coated with collagen and tissue thromboplastin at shear rates of 1100 and 110 s−1. Thrombus formation in the capillary was quantified by monitoring flow pressure changes. The intervals to 5 kPa (T5) and 40 k Pa (T40) reflect the onset and growth of thrombus formation respectively. Furthermore, fibrin and platelets in thrombi were quantified by immunostaining. T5 at both shear rates were similar in FVIII−/− and FVIII+/+ mice. T40 of FVIII−/− mice (1569 ± 565 s) was significantly delayed compared with FVIII+/+ mice (339 ± 78 s) at 110 s−1 (P < 0.05), but not at 1100 s−1. The delay was normalized by adding human FVIII (2 IU mL−1). Similarly, adding anti-FIXa aptamer to human blood prolonged T40 at 110 s−1 (P < 0.01), but not at 1100 s−1. Impaired production of fibrin due to anti-FIXa aptamer at 110 s−1 was shown in the immunostained thrombus. Our perfusion experiments demonstrated that shear rates influence thrombus formation patterns in haemophilia, and that reduced activity of intrinsic tenase (FIXa-FVIIIa) becomes evident under venous shear rates.