Antibiotics are widely used to eliminate or inhibit microorganisms. Kanamycin (Kn) is one of the antibiotics used in animal feed to treat diseases and improve growth and its residues in animal products is a global concern. Kanamycin residues in animal products can cause serious side effects, such as loss of hearing, toxicity to the kidneys, and allergic reactions to the drugs. Therefore, an accurate and easy system is essential for detection of Kn. The traditional methods used in food quality are time-consuming, difficult and require expensive equipment. Therefore, developing effective methods for food safety is needed. Aptamer-based methods can overcome the disadvantages of traditional methods. In this research, a new type of biosensor based on an aptamer specific to Kn is developed. The Kn aptamer is as a molecular detection probe and its complementary DNA acts as a signal generator for amplification in Real Time PCR. In this study, the results of five concentrations of 10-10 -4 nM of complementary DNA were investigated and the optimal concentration of 10 nM with minimum Ct was selected. After optimizing the concentration of complementary DNA, three concentrations of 2.5, 5 and 10 ng ml -1 of streptavidin and five concentration of 0, 5, 10, 15 and 20 nM of aptamer were investigated and the lowest Ct for aptamer and streptavidin was obtained at a concentration of 10 nM and 1 ng ml -1 , respectively. Also, four concentrations of 0, 2.5, 5 and 10 ng ml -1 of streptavidin were studied and concentration of 5 ng ml -1 with the lowest Ct, was selected as the best concentration. Under optimal conditions, a linear detection range (standard curve) for different concentrations of Kn (5 to 5X10 -5 ) was obtained (R 2 = 0.991) and the limit of detection was 50 fgmL -1 in the range of 5 to 5X10 -5 . The specificity of this aptasensor was excellent compared with six other antibiotics (Chloramphenicol, Rifampicilin, Cefotaxime, Tetracycline, Ampicilin and Streptomycin) and perceptible Ct value change was not observed. In addition, the validity of this aptamer was appraised by identification of Kn spiked into water (0.5, 5X10 -2 and 5X10 -4 ngmL -1 ) and milk (0.5, 5X10 -3 and 5X10 -5 ngmL -1 ) and the recoveries in water and milk were in the range of 96% and 110%. Therefore, this method indicates an inexpensive, very sensitive and specific for Kn detection and can be used with great ability for detection of a wide range of target analytes in a single PCR tube. Keywords: Kanamycin, Aptasensor, Real Time PCR, Food security