Circulating microRNAs (miRNAs) have the potential to become reliable and noninvasive biomarkers for ovarian cancer (OC) diagnosis; however, the conventional miRNAs detection techniques exhibit enduring limitations of low sensitivity and specificity. Graphene oxide (GO), a novel nanomaterial, is at the forefront of material design for extensive biomedical applications. Owing to the excellent water affinity and single-stranded DNA (ssDNA) adsorption characteristics of GO, we designed and developed a GO-based qRT-PCR assay for the detection of miRNAs associated with OC. In the GO-based qRT-PCR system, GO could significantly improve the sensitivity and specificity of the qRT-PCR assay by noncovalently interacting with primers and ssDNA and reducing the occurrence of non-specific amplification. Moreover, the detection of miRNAs associated with OC confirmed that GO-based qRT-PCR assay could differentiate benign ovarian tumors from OC (sensitivity, 0.91; specificity, 1.00). Collectively, these findings provide robust evidence that GO-based qRT-PCR assay can be effectively used as a promising method to detect miRNAs for the screening of OC patients. Display omitted •The graphene oxide-based qRT-PCR assay was used to determine miRNAs in plasma.•The graphene oxide-based qRT-PCR assay lowered the detection limit and eliminated non-specific amplification.•High sensitivity and specificity diagnostic method of ovarian cancer was established by the graphene oxide-based qRT-PCR.