Electroactive actuators based on conductive polymers currently have attracted a great deal of attention. In this study, a nanofibrous structure of polypyrrole (PPy) was used to fabricate an electroactive bending actuator. For this purpose, polyurethane/PPy (PU/PPy) nanofibrous bending actuator was fabricated through the combined use of electrospinning and in-situ chemical polymerization. The response surface methodology (RSM) was considered to find the optimal electrospinning conditions for producing PU nanofibers with the minimum diameter. The in-situ chemical polymerization method was then used to prepare a conductive layer of PPy on the surface of optimum electrospun nanofibers with p-toluenesulfonate (pTS) as the dopant. The coated nanofibers were used in the fabrication of PU/PPy-pTS nanofibrous bending actuator. The morphology and electrical, thermal, electrochemical, and electrochemomechanical properties of the fabricated actuator were investigated. By using optimum conditions of electrospinning, PU nanofibers were obtained with a diameter of 221 nm. The results showed that the produced PU/PPy-pTS nanofibers enjoy good thermal stability and have an electrical conductivity of about 276.34 S/cm. The obtained cyclic voltammetric and dynamo-voltammetric responses showed that the dominant mechanism of actuation in the fabricated PU/PPy-pTS nanofibrous actuator is the exchange of perchlorate anions with a partial exchange of lithium cations in 1M lithium perchlorate electrolyte solution. The fabricated actuator was capable of undergoing 141°r eversible angular displacement during a potential cycle. The results demonstrated that, given high porosity, large specific surface area, flexibility, and desirable electrical properties, PU/PPy nanofibrous electroactive actuator provides a lot of potential for developing artificial muscle applications.