Single-stranded DNA (ssDNA) aptamers specific to streptomycin were screened and identified from a random oligonucleotides library by affinity magnetic beads-based SELEX. After eight rounds of selection, 16 ssDNA with different sequences were identified. Then the dissociation constants (Kd) of these ssDNA were determined and an aptamer (STR1) with highest affinity for streptomycin was identified. Further study showed that aptamer STR1 exhibits very low affinity for other aminoglycoside antibiotics, indicating high specificity. With this aptamer, detection of streptomycin was achieved by using gold nanoparticles (AuNPs)-based colorimetric method. In the presence of streptomycin, the competitive binding of the target and the aptamer decreases the stability of AuNPs in NaCl solution, triggers the aggregation, and exhibits visible color change of AuNPs solution. Through UV-visible spectroscopic quantitative analysis, streptomycin can be detected in the range of 0.2-1.2 μM. The presence of other aminoglycoside antibiotics shows neglectable disturbance. Furthermore, the established method was utilized to detect streptomycin in honey, and the same low detection limit and linear detection range were achieved. Copyright © 2013 Elsevier B.V. All rights reserved.