Ovarian tumors/cancers are threatening women's health worldwide, which demands high-performance detection methods and accurate strategies to effectively detect, diagnose and treat them. Here, we report a nanographene oxide particle-functionalized microfluidic fluorescence biosensor to simultaneously detect four biomarkers, CA125, HE4, CEA and APF, for ovarian tumor/cancer diagnosis. The developed biosensor exhibits good selectivity and a large biomarker detection range with a limit of detection of 0.01 U/mL for CA125 and ~1 pg/mL for HE4, CEA and APF. The current results indicate that (1) the proposed biosensor is a promising tool for the simultaneous detection of multiple biomarkers in ovarian tumors/cancer and (2) CA125 and HE4 are strong indicators, AFP may be helpful, and CEA is a weak biomarker for ovarian tumor/cancer diagnosis. The proposed biosensor would be a potential tool, and an analytical approach for the simultaneous detection of multiple biomarkers will provide a new strategy for the early screening, diagnosis and treatment of ovarian tumors/cancers, as well as other cancers.