In this article, Ag2S quantum dots (QDs) were prepared by a facile aqueous synthesis method, using thiourea as a new sulfur precursor. Based on electrostatic interactions, 5-fluorouracil (5-Fu) was combined with the aptamer of CA125 antigen to fabricate aptamer/5-Fu complex. The surface of as-prepared Ag2S QDs was modified with polyethylenimine, followed by combination with the aptamer/5-Fu complex to form Ag-2S QDs/aptamer/5-Fu hybrids. During the combination of Ag2S QDs with aptamer/5-Fu complex, near-infrared (NIR) photoluminescence (PL) of QDs (peaked at 850 nm) was markedly reduced under excitation at 625 nm, attributed to photo-induced electron transfer from QDs to 5-Fu. However, the addition of CA125 induced obvious NIR PL recovery, which was ascribed to the strong binding affinity of CA125 with its aptamer, and the separation of aptamer/5-Fu complex from the surface of QDs. Hence, the Ag2S QDs/aptamer/5-Fu hybrids were developed as a novel NIR PL turn-on probe of CA125. In the concentration range of [CA125] from 0.1 to 106 ng mL(-1), there were a good linear relationship between NIR PL intensities of Ag2S QDs and Log[CA125], and a low limit of detection of 0.07 ng mL(-1). Experimental results revealed the highly selective and sensitive NIR PL responses of this probe to CA125, over other potential interferences. In real human body fluids, this probe also exhibited superior analytical performance, together with high detection recoveries.