Streptomycin is a type of aminoglycoside used in treatment of several infections and with serious side effects in humans and animals, such as ototoxicity and nephrotoxicity. In the present study, we focused on the preparation of streptomycin‐imprinted cotton fiber for rapid, sensitive and selective quantification of streptomycin in honey samples. The imprinted cotton fibers were characterized by attenuated total reflectance‐Fourier transform spectroscopy, scanning electron microscopy, and x‐ray photoelectron spectroscopy. In addition, the imprinted cotton fibers showed high adsorption capacity (95.4 mg/g) with an imprinting factor of 3.69 and had satisfactory regeneration ability of up to 10 regenerations without significant change in adsorption capacity. The imprinted cotton fibers showed good linearity with a correlation coefficient of 0.9989 and limit of detection was determined to be 0.29 ng/mL for streptomycin in honey samples. The proposed method showed acceptable recovery ranges (98.0%–100.3%) with lower inter‐day and intra‐day precisions. Moreover, the performance of the proposed method was compared to the ELISA method. The results showed that the present method is as sensitive and selective as ELISA. It is concluded that the imprinted cotton fibers could be a potential alternative to antibiotic quantification based on traditional immunoassays or chromatographic techniques.