Background: In this work, an aptamer-based biosensor was successfully developed based on the salt-induced gold nanoparticle (AuNP) aggregation phenomenon for the detection of carbohydrate antigen 125 (CA125), which is an important tumor marker for ovarian cancer. Materials and Methods: Citrate-coated AuNPs are relatively highly dispersed NPs. In the presence of different salts, the electrostatic stability of NPs is reduced, and depending on the type of salt and its concentration, different degrees of aggregation occur. On the other hand, the aptamer is easily adsorbed on the AuNP surface and can prevent salt-induced AuNP aggregation. This phenomenon was used in this study to develop a simple biosensor for the detection of CA125. Results: In the presence of CA125, the aptamer was desorbed from the AuNP surface to bind to its antigen due to the higher affinity, leading to the aggregation of AuNPs and a change in the absorption spectra of the solution. Under the optimum condition, the fabricated aptasensor showed a linear range of 15-160 U/mL with a limit of detection (LOD) of 14.41 U/mL. Conclusion: The aptasensor exhibited good repeatability with notable selectivity with regard to CA125 detection, even in human serum samples, as compared to the enzyme-linked immunosorbent assay (ELISA). In conclusion, the engineered aptasensor can serve as a promising tool for the simple, rapid, and cost-effective detection of CA125.