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In this work, a rapid, sensitive and selective sensor was designed based on electrospun carbon nanofibers-modified screen printed electrode (CNFs/SPE) for the determination of tramadol. CNF based membrane was prepared through electrospinning of polyacrylonitrile followed by stabilization and carbonization. The as-prepared CNF membrane was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Raman spectroscopy to investigate its morphology and carbon structure. The electrical conductivity of CNFs sheet was determined using 4-point probes electrical impedance measuring technique. The SPE surface was modified by CNFs membrane and the CNFs/SPE was used for qualitative and quantitative determination of tramadol using cyclic and square wave voltammetry techniques, respectively. Several parameters including electrochemical behavior of the unmodified and modified electrodes, the type and concentration of the supporting electrolyte, the pH value of buffer solution, and the effect of scan rate were examined. Modification by CNFs considerably improved the electrochemical response of the sensor. Under the optimized conditions, a linear range of 0.05–100 nM was obtained and the limits of quantification and detection were calculated to be 0.05 nM and 0.016 nM, respectively. Likewise, repeatability, reproducibility and stability of the proposed electrode were evaluated to check the applicability of the developed platform. Finally, the developed sensor was applied for the determination of tramadol in the urine sample. The results demonstrated the applicability of the sensor for accurate detection of tramadol in real samples.