Continuous polyacrylonitrile nanofiber yarns were fabricated by the homemade multiple conjugate electrospinning apparatus, and the principle of yarn spinning was studied. The effects of the applied voltage, flow rate, spinning distance, and funnel rotary speed on the diameter and mechanical properties of nanofiber yarn were analyzed. The diameter of the nanofibers decreased with increasing applied voltage and the flow rate ratio of the positive and negative needles (FP/FN), whereas the diameter of nanofibers increased with increasing overall flow rate and needle distance between the positive and negative. Subsequently, the diameter of the yarns increased first and then decreased with increasing applied voltage, FP/FN, and needle distance. However, the diameters of the yarns increased dramatically and then remained stable with increasing overall flow rate. The nanofibers were stably aggregated and continuously bundled and then uniformly twisted into nanofiber yarns at an applied voltage of 20 kV, an overall flow rate of 6.4 mL/h, a needle distance of 18.5 cm, and an FP/FN value of 5:3. With increasing funnel rotary speed, the diameters of the nanofibers and yarns decreased, whereas the twist angle of the nanofiber yarns gradually enlarged. Meanwhile, an increase in the twist angle brought about an improvement in the yarn mechanical properties. Nanofiber yarns that prepared showed diameters between 70 and 216 μm. Nanofiber yarns with a twist angle of 65° showed a tensile strength of 50.71 MPa and an elongation of 43.56% at break, respectively. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40137.