Despite the relatively low transfection efficiency and low specific foreign protein productivity (q(p)) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher q(p) attributable to EBNA-1 but not PyLT expression. The q(p) for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased q(p). Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.