Objective To investigate the expression of ecotropic virus integration site 1 (EVI1) and karyopherin-α2 (KPNA2) in three negative breast cancer (TNBC), and their relationship with the clinicopathological characteristics and prognosis. Methods Seventy three paraffin samples of cancer tissues and paracancerous tissues from TNBC patients and 70 normal breast tissue (control group) from January 2012 to January 2015 were collected. The expression of EVI1 and KPNA2 were detected by immunohistochemistry. The relevant clinicopathological data were collected, and the correlation between the expression of EVI1, KPNA2 and the clinicopathological parameters of TNBC were analyzed. Kaplan-Meier survival analysis was used to analyze the difference of progression free survival (PFS) and overall survival (OS) of TNBC patients under different expression of EVI1 and KPNA2. Results The positive expression rates of EVI1 and KPNA2 in the cancer tissues, adjacent tissues and the control group were statistically significant (P < 0.05). The positive expression rates of EVI1 and KPNA2 in TNBC cancer tissues were higher than those in the adjacent tissues and the control group (P < 0.05), while the difference in the positive expression rates of EVI-1 and KPNA2 in the adjacent tissues and the control group was not statistically significant (P > 0.05). The positive expressions of EVI1 of TNBC patients with the different histological grade, lymph nodes and tumor thrombus were significantly different (P < 0.05), and the expression of KPNA2 between TNBC patients with lymph nodes metastasis or not were significantly different (P < 0.05). The results of Kaplan-Meier survival analysis showed that the survival rate of PFS and OS in patients with positive expression of EVI1 and KPNA2 were lower than those in patients with negative expression (P < 0.05). Conclusion The positive expression of EVI1 and KPNA2 are related to the malignant invasion and poor prognosis of TNBC patients. Valuating the expression status of EVI1 and KPNA2 can provide a certain basis for the prognosis prediction of TNBC patients.