8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) is a crucial biomarker of oxidative DNA damage. Herein, a highly sensitive fluorescent aptasensor for detecting 8-oxo-dG in urine was developed based on aptamer-mediated quenching of amino-terminated oligo(ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) toward green emissive carbon dots (G-CDs). G-CDs were employed as fluorometric reporters. Upon addition of 8-oxo-dG, it formed a complex with an anti-8-oxo-dG aptamer, resulting in the quenching of fluorescence as NH2-TEG-AuNPs absorbed the emission from G-CDs. Initially, NH2-TEG-AuNPs suppressed the fluorescence intensity of G-CDs via the inner-filter effect (IFE). With the addition of the aptamers to a mixture of G-CDs and NH2-TEG-AuNPs, the electrostatic interactions between the aptamer and NH2-TEG-AuNPs resulted in the aggregation of NH2-TEG-AuNPs and the recovery of the fluorescence intensity of the quenched G-CDs. NH2-TEG-AuNPs dispersed due to the high affinity between the aptamer and 8-oxo-dG molecules, and the system demonstrated a fluorescence ″turn-off″ response of G-CDs. Thus, it was possible to determine the 8-oxo-dG concentration. A fluorescence calibration curve was constructed with two linear ranges: 100–2000 and 2000–10,000 nM. The limit of detection (LOD) for 8-oxo-dG was 15.89 nM. This approach was successfully applied to assess 8-oxo-dG in synthetic urine samples with mean recoveries ranging from 99 to 120% and relative standard deviations (RSDs) between 1.1 and 4.1%. The analytical performance was comparable to a commercially available enzyme-linked immunosorbent assay (ELISA) kit. This proposed fluorescent aptasensor for 8-oxo-dG detection in urine exhibited high selectivity and sensitivity over a broad concentration range against the target molecule and has the potential to be developed as a new platform for rapid screening of 8-oxo-dG.