Abstract: Designing and fabricating flexible strain sensors with simultaneous high sensitivity and a broad linear working region remains a considerable challenge. In this work, thermoplastic polyurethane (TPU) fiber films as flexible substrate were first prepared via electrospinning. Then, the carbon nanotubes (CNT) were anchored onto the surface of a TPU fiber films via ultrasonication as the driving force. Finally, the polypyrrole (PPy) conductive layer were grown on the surface of CNT/TPU by a self-assembled process, and PPy-CNT/TPU composites with double conductive layers were obtained. The morphology and mechanical properties of TPU, CNT/TPU and PPy-CNT/TPU were analyzed. The sensing mechanism and strain response characteristics of flexible strain sensor based on PPy-CNT/TPU are investigated. The results indicated that the PPy-CNT/TPU strain sensor has a Gauge Factor (GF) of 140.9 in a strain ranges from 0 to 180% and GF of 438.7 in a strain ranges from 180% to 400%. In addition, the strain sensor has a response time of 40 ms and a recovery time of 69 ms as well as superior cyclic stability over 6000 cycles. The strain sensor has been successfully used to monitor human movements’ signals, which indicates great potential for wearable electronics and human-machine interfaces application.