A simple ultrasonic energy assisted synthesis of β-cyclodextrin (β-CD) supported carbon nanofiber composite (CNF) and its potential application in electrochemical sensing of antibiotic nitrofurantoin (NFT) is reported. The elemental composition and surface morphology of the β-CD/CNF composite was validated through Field emission scanning electron microscopy, energy dispersive X-ray microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis. The uniform enfolding of hydrophilic β-CD over CNF enhance the aqueous dispersion and offer abundant active surface to the β-CD/CNF composite. Further, the electrocatalytic efficacy of the β-CD/CNF composite is utilized to fabricate an electrochemical sensor for the high sensitive quantitative detection of NFT. Under optimized analytical conditions, the sensor displays a broad working range of 0.004-308 µM and calculated detection limit of 1.8 nM, respectively. In addition, the sensor showcased a good selectivity, storage, and working stability, with amiable reproducibility. The point-of-care applicability of the sensor was demonstrated with NFT spiked human blood serum and urine sample with reliable analytical performance. The simple, cost-effective NFT sensor based on β-CD/CNF offered outstanding analytical performance in real-world samples with higher reliability.