In this study, a new magnetic dual-template imprinted polymer modified with functionalized SBA-15 and graphene oxide (Fe3O4@SBA-15-NH-GO/dtMIP) was synthesized using surface imprinting technique. The synthesized polymer was used for preconcentration of amitriptyline and nortriptyline in plasma of mice (male BALB / cJRj mice) using µ-solid phase extraction (µ-SPE) and simultaneous determination by HPLC technique. The mice plasma was studied at regular intervals after taking two drugs. The polymer was characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray energy diffraction spectroscopy (XRD), vibrating sample magnetometer (VSM), and Nitrogen adsorption/desorption analysis. By studying the response surface methodology (RSM) and desirability function (DF), the optimal experimental condition was determined as 16.72 min for ultrasonic time, 8.11 for pH and 40.51 mg for polymer dosage. The results of the analysis were reported for amitriptyline in mice plasma in the concentration range 6.0–950.0 and the detection limit was achieved as 1.5 ng mL−1 respectively. For nortriptyline the LDR and LOD was reported as 5.0–850.0 and 1.1 ng mL−1 respectively.