Objective To establish an immortalized tree shrew corneal stromal cell line and to study the virus infection. Methods The primary corneal stromal cells (CSCs) of tree shrew were isolated and cultured by tissue block adhesion method. CSCs were transfected a lentivirus carrying SV40T gene, monoclonal clones were selected for passage culture. The characteristics of CSCswere tested through morphological observation、immunofluorescence、Karyotypeand cell proliferation curve. Using Herpes simplex virus-1(HSV-1)、ZIKV(GZ01 strain)、Dengue virus typeⅡand H1N1 A/Puerto Rico/8/1934(A/PR/8)to infect the CSCs. Virus titers were detected by CCDI50. Results the immortalized tree shrews CSCs transmitted to more than 50 passages were spindle-shaped and had good cell morphology and structure. High level of Vimentin and SV40T were detected by immunofluorescence. The cell growth curve showed the cells were in the logarithmic phase on days 4-5, and grew vigorously. The number of chromosomes of the primary cell karyotype was stable at62, while immortalized CSCs was 64 at P21 and P56. The results of virus titer showed that the immortalized tree shrew CSCs were sensitive to HSV-1、ZIKV(GZ01 strain)、Dengue virus typeⅡand H1N1 A/Puerto Rico/8/1934(A/PR/8),and the virus titers were 1.32×105^TCID50/mL、5.62×106^TCID50/mL、2.69×107^TCID50/mL、7.76×104^TCID50/mL respectively. Conclusions The immortalized tree shrew CSCs were successfully established, suggesting that the cell line can be used in study of the mechanism of herpes simplex virus, Zika virus, Dengue virus and influenza A virus infection of corneal diseases and antiviral drugs.