Triboelectric nanogenerators (TENGs) are promising devices for capturing mechanical energy. However, traditional polymer triboelectric materials result in a burden to the environment, and the natural/biodegradable tribo-materials have the disadvantage of poor output performance. For this purpose, we proposed a polyethylene oxide (PEO)/cysteine composite nanofiber film (PCF) which is prepared from biodegradable polymer PEO and natural cysteine. Thanks to the superior tribo-positive properties of PEO and cysteine, the electrical performance of a PCF-based TENG (PC-TENG) with 4 wt% cysteine is several times greater than that of a pure PEO nanofiber film. In addition, the PC-TENG exhibits better power density (6.6 W m−2), which is 3–110 times more than that in studies using related eco-friendly materials as the tribo-layer. Importantly, we designed a multi-layer funnel-shaped TENG (MF-TENG) constructed from 4 layers of PC-TENG, which can effectively harvest tiny mechanical energy to build self-powered electronic devices by integrating a power management circuit. This research offers an efficient approach for the practical application of natural and environmental-friendly material-based TENGs in energy harvesting and power supply in the Internet of Things.