In this study, a novel sample preparation technique coupled with liquid chromatography was developed to determine the trace residues of antibiotics in food samples. Target analytes were extracted by accelerated solvent extraction (ASE), followed by the concentration and cleanup using micro-solid-phase extraction (μ-SPE). Variables affecting the ASE and μ-SPE were optimized to achieve the maximum extraction efficiency, besides the performance of the developed method was evaluated. Low detection limits of 7.4–16.3 ng/g and low quantification limits of 24.7–53.8 ng/g were achieved under optimized conditions. The recoveries of antibiotics ranged from 92 to 105 % with relative standard deviation of less than 9.3 %. The predominance was showed when compared to conventional ASE and μ-SPE. According to the results, ASE-μ-SPE was proved to be a simple and effective sample preparation method for the analysis of trace organic contaminants in food samples.