A novel sample preparation method for auxin analysis in plant samples was developed by vacuum microwave-assisted extraction (VMAE) followed by molecularly imprinted clean-up procedure. The method was based on two steps. In the first one, conventional solvent extraction was replaced by VMAE for extraction of auxins from plant tissues. This step provided efficient extraction of 3-indole acetic acid (IAA) from plant with dramatically decreased extraction time, furthermore prevented auxins from degradation by creating a reduced oxygen environment under vacuum condition. In the second step, the raw extract of VMAE was further subjected to a clean-up procedure by magnetic molecularly imprinted polymer (MIP) beads. Owing to the high molecular recognition ability of the magnetic MIP beads for IAA and 3-indole-butyric acid (IBA), the two target auxins in plants can be selectively enriched and the interfering substance can be eliminated by dealing with a magnetic separation procedure. Both the VMAE and the molecularly imprinted clean-up conditions were investigated. The proposed sample preparation method was coupled with high-performance liquid chromatogram and fluorescence detection for determination of IAA and IBA in peas and rice. The detection limits obtained for IAA and IBA were 0.47 and 1.6 ng/mL and the relative standard deviation were 2.3% and 2.1%, respectively. The IAA contents in pea seeds, pea embryo, pea roots and rice seeds were determined. The recoveries were ranged from 70.0% to 85.6%. The proposed method was also applied to investigate the developmental profiles of IAA concentration in pea seeds and rice seeds during seed germination.