It is now well accepted that the quantification of anticancer drugs in biological fluids is very important. For the first time, novel hyperbranched dendrimers grafted on SiO2 nanoparticles were synthesized as nanosorbents for trace extraction of exemestane. SiO2 nanoparticles modified by (3-aminopropyl) triethoxysilane were grafted with melamine/glutaraldehyde dendrimers (four generations) and characterized via FT-IR, FE-SEM equipped with EDS. First, to reduce the effects of the matrix, the synthesized nano-adsorbent has been used for solid phase extraction of exemestane in aqueous solution, and the parameters affecting the adsorption efficiency including pH, contact time and temperature, have been optimized by Box–Behnken design. In the next step, the loaded drug was ultrasonically desorbed and used as a donor phase in the HF-LPME system in order to preconcentrate and reduce the detection limit of the method. The type of extraction solvent and the amount of pH, time, temperature and ionic strength are optimized in the HF-LPME stage. A detection limit of 0.1 μg L−1 with a linear range of 0.3–300 μg L−1 was obtained. The adsorption isotherm results fitted well with the Langmuir isotherm model (R2 > 0.9966). The proposed method along with high-performance liquid chromatography was successfully used to determine exemestane in biological and pharmaceutical samples with a recovery of 92.5–97.7 %.