In serum, the most promising liquid biopsy biomarkers were c-Maf inducing protein (CMIP), glutamate decarboxylase 1 (GAD1), nucleoside diphosphate kinase 1 (NME1), CDP-diacylglycerol synthase 1 (CDS1), and cyclin-dependent kinases regulatory subunit 1 (CKS1B), showing AUC values of 0.957, 0.928, 0.899, 0.893, and 0.891, respectively, for the diagnosis of CCA in comparison with control group while the combination of CMIP, NME1 and CKS1B provided the maximum diagnostic capacity (AUC: 1.000). The gene discussed is GAD1; the disease is cholangiocarcinoma.