Isolation conditions for five pesticides (metazachlor, tebuconazole, λ-cyhalothrin, chlorpyrifos, and deltamethrin) from rape oil samples were examined using the dispersive solid-phase graphene extraction technique. To determine the optimal extraction conditions, a number of experimental factors (amount of graphene, amount of salt, type and volume of the desorbing solvent, desorption time with and without sonication energy, and temperature during desorption) were studied. The compounds of interest were separated and detected by an HPLC-UV employing a Kinetex XB-C18 column and a mobile phase consisting of acetonitrile and water flowing in a gradient mode. The optimized extraction conditions were: the amount of graphene 15 mg, desorbing solvent (acetonitrile) 5 mL, time desorption 10 min at 40°C, and amount of NaCl 1 g. The detection limit for metazachlor, tebuconazole, λ-cyhalothrin, and chlorpyrifos was 62.5 ng·g-1, and for deltamethrin, it was 500 ng·g-1. The obtained results lead to the conclusion that graphene may be successfully used for the isolation of the five pesticides from rape oil. However, their determination at low concentration levels, as they occur in real oil samples, requires the employment of appropriately highly sensitive analytical methods, as well as a more suitable graphene form (e.g., magnetically modified graphene).