Polyurethane (PU) was synthesized from polypropylene glycol (PPG 2000) and diphenylmethanediisocyanate (MDI-50) by prepolymerization method. Carbon nanotubes/polyurethane (CNT/PU) was obtained by adding carbon nanotubes in the prepolymerization process, while carbon black/polyurethane (CB/PU) was obtained by adding carbon black in the process of chain extension. Then, CNT/PU nanofiber and CB/PU nanofiber were obtained by electrospinning technique. Two nanofibers were characterized by SEM, TEM, FTIR, DSC and XRD. The electrical, mechanical and sensing properties of nanofiber membranes were tested. The results showed that the degree of microphase separation of the prepared polyurethane nanofibers increased/decreased due to the selective embedding of CNT/CB in the soft and hard segments of polyurethane. Compared with that of pure PU nanofiber membrane, the elongation at break of CNT/PU nanofiber membrane decreased, and the breaking strength of CB/PU nanofiber membrane increased. Herein, the content of conductive filler (CNT, CB) was based on the mass of polyurethane (total mass of PPG 2000, MDI-50 and anhydrous ethylenediamine). When the content of CNT was 6.0%, the prepared CNT/PU nanofiber membrane had a resistivity of 1.22 Ω•m and sensing coefficient (GF) of 45.64 (tensile strain in 80%~120%). While the content of CB was 12.5%, the resistivity and GF of the prepared CB/PU nanofiber membrane were 0.14 Ω•m and 167.43 (tensile strain in 80%~120%), respectively. © 2020, Editorial Office of FINE CHEMICALS. All right reserved.