Electrospun triboelectric nanogenerators (TENGs) are favored in flexible, wearable devices. In this article, the positive friction layers of TENGs with biomimetic strawberry structures were efficiently prepared using free-surface electrospinning technology. The dielectric constant of multistrawberry-structured (MSS) micro/nano fibers with the highest specific surface area was increased by about 1.5 times, significantly enhancing the charge-trapping ability of positive friction layers. Subsequently, the effects of fiber microstructure, contact area, membrane thickness, external force, air gap, and frequency on the output properties of MSS-TENG were explored. The optimal device performed well at 25 N and 3 Hz, with an open-circuit voltage of 301.93 V, a short-circuit current of 7.34 μA, a transfer charge of 103.89 nC, and a power density of 0.617 W/m2, lighting up 300 light-emitting diodes and driving portable electronic devices to operate normally. Moreover, MSS-TENG could maintain stable output after nearly 40 min of cycling.