Graphene oxide (GO)-based dispersive solid phase extraction (D-SPE) method combined with multi-step preparation has been put forward for the evaluation of trace ochratoxin A (OTA) in chicken liver. The homogenized tissue sample was initially clean-up and extracted with petroleum ether, ethyl acetate and 0.1 mol/L sodium bicarbonate solution and then adsorbed using GO originally targeting for OTA. The adsorbed OTA was then eluted twice by 1 mL of methanol and finally quantified by high performance liquid chromatography fluorescence detection (HPLC-FD). As a result, the matrix effect of chicken liver tissue was removed to a great extent after a series of processing steps. Under the optimum conditions, the functionality of the method was attested and this method demonstrated proper sensitivity, with detection and quantification limits equal to 0.02 ng/mL and 0.04 ng/mL, respectively. The method turned out to be accurate with recoveries ranging from 86.4 to 102.9%. Furthermore, the established method was applied to assess OTA contamination in different batches chicken liver, which revealed that two supermarket-sold and three individual domestic samples were contaminated with OTA (33% frequency) and the proposed method was suitable for continuous monitoring for OTA in chicken liver to ensure food safety.