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Nanofiber yarns are important building blocks for making three-dimensional nanostructures, e.g. through a knitting or weaving process, with better mechanical properties than nanofiber nonwovens and well-controlled fibrous construction. However, it still remains challenging to produce quality nanofiber yarns in a sufficient rate. In this study, we have proven that online stretching during electrospinning of nanofiber yarns can considerably improve fiber alignment and molecular orientation within the yarn and increase yarn tensile strength, but reduce fiber/yarn diameters. By compensating twist during online stretching, the device can prepare nanofiber yarns with different stretch levels, but maintaining the same twist multiplier. This allows us to examine the effect of stretching on fiber and yarn morphology. It was interesting to find that on increasing the stretching ratio from 0% to 95%, the yarn diameter reduced from 135.1 ± 20.3 μm to 46.2 ± 10.2 μm, and the fiber diameter reduced from 998 ± 141 nm to 631 ± 98 nm, whereas the yarn tensile strength increased from 48.2 ± 5.6 MPa to 127.7 ± 5.4 MPa. Such an advanced yarn electrospinning technique can produce nanofiber yarn with an overall yarn production rate as high as 10 m min−1. This may be useful for production of nanofiber yarns for various applications.