A graphene-based solid phase extraction column was prepared for the isolation of tetracyclines from milk followed by determination by high performance liquid chromatography. Graphene provided better separation for tetracyclines than amine modified graphene and carboxyl modified graphene. The optimized graphene-based solid phase extraction column showed high absorption capacities (greater than 4,660 ng) and high recoveries (exceeding 92%) for tetracycline, oxytetracycline, chlortetracycline, and doxycycline and was successfully reused at least fifty times. The limits of detection in milk were from 10 to 20 ng/mL, with recoveries between 82.3 and 103.6%. Furthermore, the system showed superior performance than two commercial solid phase extraction cartridges with respect to recovery, purification, and reusability. Therefore, this approach is suitable for the determination of tetracyclines in milk.