The T-box 3 (TBX3) transcription factor has been shown to serve multiple roles in normal development. Recent findings have revealed that TBX3 is overexpressed in different types of carcinomas, including breast, cervical, ovarian, melanoma, pancreatic, lung, liver, bladder, head and neck. Therefore, the present study investigated the significance of TBX3 as a diagnostic marker of breast cancer. To achieve this aim, breast cancer samples and their adjacent normal tissues were collected from 51 breast cancer patients from the European Gaza hospital during 2015-2016. Sections from each sample were immune-stained by anti-TBX3 and suitable secondary and tertiary antibodies. TBX3 levels were evaluated in cancerous and normal samples. Clinicopathological data for each patient were documented. The correlation between TBX3 levels and the clinicopathological parameters were statistically tested. The results revealed that TBX3 is significantly overexpressed in breast cancer tissues when compared with normal tissues. Furthermore, TBX3 was mainly a cytoplasmic protein in normal and breast cancer tissues. Notably, TBX3 levels exhibited a sensitivity of 78.4%, specificity of 79.6%, accuracy of 79% and area under the curve of 0.791 (0.700-0.882) at a cutoff value=9 as breast cancer marker. However, no significant associations were observed between TBX3 levels and other breast cancer markers including oestrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, cancer antigen 15-3 and breast cancer stages. Altogether, these results suggested that TBX3 overexpression may be a potential biomarker for breast cancer.