Identification of proteins as biomarkers for early detection of cancer and therapeutic targets for cancer treatment are important issues in precision medicine. Oral cavity squamous cell carcinoma (OSCC) is a common cancer worldwide and represents a serious and growing problem in many parts of the globe, including Taiwan. To help early detection of OSCC, our group have put efforts in the past decades to discover/verify potential protein biomarkers using saliva, a kind of protein-rich body fluid that directly contacts the oral lesions, and we developed a four-protein marker panel, which may be a clinically effective tool for detecting OSCC and monitoring high-risk oral potentially malignant disorders (Yu et al., PNAS USA, 2016 Oct 11;113(41):11549-11554). Here, we attempted to screen DNA aptamers toward these four biomarkers for further diagnosis assay development. A plated-based platform of systematic evolution of ligands by exponential enrichment (SELEX) was developed for screening DNA aptamers against one of OSCC biomarkers, kininogen-1 (KNG1). After ten rounds of SELEX, the reserved DNA pools were submitted to next-generation sequencing (NGS), and six anti-KNG1 aptamers were discovered. The KD value of these aptamers toward KNG1 can achieve 10-9 M level. Aptamers have many advantage comparing with antibodies, such as small size, stability, easy modification, low cost, and batch-to-batch consistency. Using aptamer to develop detection kits is an alternative strategy for biomarker measurement. These newly generated anti-KNG1 aptamers represent valuable tools for future development of the sandwich-ELISA assay and lateral flow strip assay.