: The detection of ovarian cancer remains challenging due to the lack of reliable serum biomarkers that reflect malignant transformation rather than mere tumor presence. We developed a novel biotest using an immortalized human fallopian tube epithelial cell line (TY), which exhibits anchorage-independent growth (AIG) in response to cancer-associated serum factors. : Sera from ovarian and breast cancer patients, non-cancer controls, and ID8 ovarian cancer-bearing mice were tested for AIG-promoting activity in TY cells. : TY cells (passage 96) effectively distinguished cancer sera from controls (68.50 ± 2.12 vs. 17.50 ± 3.54 colonies, < 0.01) and correlated with serum CA125 levels ( = 0.73, = 0.03) in ovarian cancer patients. Receiver operating characteristic (ROC) analysis showed high diagnostic accuracy (AUC = 0.85, cutoff: 23.75 colonies). The AIG-promoting activity was mediated by HGF/c-MET and IGF/IGF-1R signaling, as inhibition of these pathways reduced phosphorylation and AIG. In an ID8 mouse ovarian cancer model, TY-AIG colonies strongly correlated with tumor burden ( = 0.95, < 0.01). : Our findings demonstrate that the TY cell-based AIG assay is a sensitive and specific biotest for detecting ovarian cancer and potentially other malignancies, leveraging the fundamental hallmark of malignant transformation.