Cholangiocarcinoma (CCA), derived from the bile duct, occurs with a relatively high incidence in Northeast Thailand. Early diagnosis is still hampered by the lack of sufficient biomarkers. In recent years, biomarker discovery using secretomes has provided interesting results, including our studies on CCA secretomes, especially with three-dimensional cell cultures. Thus, cells cultured using the hollow fiber bioreactor (HFB) with 20 kDa molecular weight cut-off (MWCO) yielded higher quality and quantity of secretomes than those from conditioned media of the monolayer culture (MNC) system. In this study, we employed the HFB culture system with 5 kDa MWCO and compared conditioned media from the HFB and MNC systems using two-dimensional gel electrophoresis, followed by identifying proteins of interest by liquid chromatography and mass spectrometry (LC/MS/MS). Two out of 4 spots of NGAL or lipocalin-2 were found to show highest increase in expression of 19.93-fold and 18.79-fold in HFB compared to MNC. Interestingly, all 14 proteasome subunits including proteasome subunit α type-1 to type-7 and β type-1 to type-7 showed 2.92-fold to 12.13-fold increased expression in HFB. The protein-protein interactions of upregulated proteins were predicted, and one of the main interaction clusters involved 20S proteasome subunits. Proteasome activity in the HFB conditioned media was also found to be higher than that in MNC conditioned media. Three types of proteasome subunit were also validated by immunoblotting and showed higher expression in the HFB system compared to MNC system. Proteasome subunit α type-3 (PSMA3) showed the highest level in plasma of cholangiocarcinoma patients compared to normal and hepatocellular carcinoma patients by immunodetection, and is of interest as a potential biomarker for cholangiocarcinoma.