This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-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 and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several 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, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 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.