The von Willebrand factor (VWF) is a human plasma protein that plays a key role in the initiation of the formation of thrombi under high shear stress in both normal and pathological situations. It is believed that VWF undergoes a conformational transition from a compacted, globular to an extended form at high shear stress. In this paper, we develop and employ an approach to visualize the large-scale conformation of VWF in a (pressure-driven) Poiseuille flow of water-glycerol buffers with wide-field single molecule fluorescence microscopy as a function of shear stress. Comparison of the imaging results for VWF with the results of a control with λ-phage double-stranded DNA shows that the detection of individual VWF multimers in flow is feasible. A small fraction of VWF multimers are observed as visibly extended along one axis up to lengths of 2.0 μm at high applied shear stresses. The size of this fraction of molecules seems to exhibit an apparent dependency on shear stress. We further demonstrate that the obtained results are independent of the charge of the fluorophore used to label VWF. The obtained results support the hypothesis of the conformational extension of VWF in shear flow.