The urge for sensitive, facile, minimally invasive, and fast detection method of CA-125, a significant and crucial biomarker in ovarian malignancy, is currently substantial. This paper describes the detailed construction and characterization of a newly designed optical nano-biosensor to detect CA-125 accurately and sensitively. The fabricated sensor consists of a nano-gold thin film doped into a matrix of sol-gel, exhibiting a centered fluorescence band at 423 nm when excited at 340 nm. The quantification of CA-125 relies on its quenching ability of this fluorescence signal. The sensor was challenged to evaluate its sensitivity and specificity in detecting CA-125 present in samples collected from ovarian cancer diagnosed patients and compared to samples from healthy women as a control. Our findings revealed that the developed biosensor had a sensitivity of 97.35% and a specificity of 94.29%. Additionally, a wide linearity range over 2.0-127.0 U mL-1 for CA-125 was achieved with a detection limit of 1.45 U mL-1. Furthermore, the sensor could successfully discriminate samples between healthy and diseased people, which demonstrates its suitability in CA-125 assessment. © 2021 The Authors. Published by American Chemical Society.