This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteinsin cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes fromCCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated usinggel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed byLC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions,signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients andnon-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based andsolution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles inseveral cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis).The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein wereidentified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OVinfection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4)were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.