Antibiotics are powerful tools to treat bacterial infections, but antibiotic pollution is becoming a severe threat to the effective treatment of human bacterial infections. The detection of antibiotics in water has been a crucial research area for bioassays in recent years. There is still an urgent need for a simple ultrasensitive detection approach to achieve accurate antibiotic detection at low concentrations. Herein, a field-effect transistor (FET)-based biosensor was developed using ultraclean graphene and an aptamer for ultrasensitive tetracycline detection. Using a newly designed camphor-rosin clean transfer (CRCT) scheme to prepare ultraclean graphene, the carrier mobility of the FET is found to be improved by more than 10 times compared with the FET prepared by the conventional PMMA transfer (CPT) method. Based on the FET, aptamer-functionalized transistor antibiotic biosensors were constructed and characterized. A dynamic detection range of 5 orders of magnitude, a sensitivity of 21.7 mV/decade, and a low detection limit of 100 fM are achieved for the CRCT-FET biosensors with good stability, which are much improved compared with the biosensor prepared by the CPT method. The antibiotic sensing and sensing performance enhancement mechanisms for the CRCT-FET biosensor were studied and analyzed based on experimental results and a biosensing model. Finally, the CRCT-FET biosensor was verified by detecting antibiotics in actual samples obtained from the entrances of Bohai Bay.