The development of microfabrication and surface machining techniques has made solid surfaces more precise, and the gap dimensions in mechanical systems such as Micro Electro Mechanical Systems and Micro Total Analysis Systems have decreased from the order of micrometers to the order of nanometers. Because the fluid flow properties in nanogaps are different from those in the bulk, experimental measurement of flow velocity in nanogaps is necessary to design nanogap mechanical systems and verify the principle of their operation. In general, the lateral flow velocity distribution can be measured by mixing fluorescent particles with the fluid and tracking the particles with a microscope with high lateral and temporal resolution. However, in previous studies, quantitative measurement of flow velocity in nanogaps could not be achieved due to the influence of the interaction between the fluorescent particles and solid surfaces because the diameters of the particles were large, and ranged from tens of nm to tens of µm. The objective of this study is to quantitatively measure the lateral flow velocity distribution in nanogaps by particle image velocimetry using quantum dots with diameters in the single-digit nanometer range. We have implemented a simultaneous quantitative measurement of the nanogap shape by optical interferometry, and the flow velocity distribution by PIV using quantum dots. Focusing on the squeeze flow caused by narrowing the gap, we have shown that PIV using quantum dots can achieve quantitative measurement of velocity distribution in 100 nm-level-gaps.Graphical Abstract: