Ovarian cancer is a significant and life-threatening malignancy among women worldwide. Selective tumor targeting by molecular agents is essential for the development of effective therapeutics and diagnostics. Recognizing the disparities between in vivo and in vitro environments, we have implemented an in vivo SELEX strategy to identify DNA aptamers capable of specifically localizing to ovarian tumors in situ. Aptamer L1 has emerged as a promising candidate with a high binding affinity to Insulin-like Growth Factor Binding Protein 3 (IGFBP3), a tumor-promoting protein overexpressed in ovarian cancer. Notably, aptamer L1 effectively downregulates IGFBP3 expression and suppresses ovarian cancer cell proliferation. The unique molecular characteristics of aptamer L1 underscore its potential as a theranostic agent for ovarian cancer.