Nowadays, flexible triboelectric sensors are the most promising candidates for wearable energy harvesting and medical applications. In the present investigation, the self-powered flexible triboelectric sensors (SF-TESs) were fabricated by using roughness-created Ecoflex cast film and melt-blown nonwoven polyurethane layers. SF-TESs were prepared using sandpaper (60 grit size) as a substrate, showing better triboelectric performance due to high surface roughness and low density compared to others, and it is an optimum condition. The optimized SF-TES can scavenge a maximum open-circuit output voltage (V OC) and power density values for the sensor without and with a spacer were 139 V and 1.6 Wm–2 and 320 V and 6 Wm–2, respectively. The spacer sample showed 2.3 times higher V OC and 3.7 times higher power density values than without a spacer at a constant load of 4.4 N and a frequency of 5 Hz. The SF-TES can directly operate 15 light-emitting diodes (LEDs) and switch on an LCD timer, and it is powered for 5 s when connected through a rectifier and a capacitor. In contrast, the sensor with a spacer can operate more than 100 LEDs directly. Further, the triboelectric output performances V OC and short-circuit current (I SC) gradually increase from 65 to 139 V and 2.3 to 4.6 μA, respectively, when the external load increases from 2.2 to 8.8 N. Based on the above results, we can clearly say that the surface roughness, spacing between the frictional layers, and external load are critical parameters for enhancing the triboelectric performance. The as-prepared SF-TESs have potential medical applications in the intelligent beds for observing the physical activity of coma patients, patients suffering from cardiovascular diseases, and sleep apnea.