Background: Breast cancer (BC) is one of the most common non-skin cancers in women and the fifth most common cause of cancer death worldwide. Both the prognosis and survival rate of BC patient improve considerably if the disease is discovered at an early stage. Methods: In the present study, we analyzed qualitatively and quantitatively volatile organic compounds (VOCs) in the headspace over urine, blood and tissue samples to identify VOCs characteristic for diagnosing BC. The study comprised 150 women with non-metastatic BC Stage II and an equal number of age-matched Healthy Controls (HC). Collected urine, blood and BC tissue samples were analyzed using the Electronic Nose (E-Nose) and Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) techniques. Results: BC was directly related to E-Nose responses for urine, blood and tissue samples. Linear Discriminant Analysis showed separate clusters for urine, blood and tissue sample for BC patients and HC participants, where the first two principal components explained more than 98.84% of the variance in signals with no false-positive (HC participants) or false-negative (BC patients) results. Conclusions: SIFT-MS showed the expression of 10 aldehydes in tissue specimens and blood samples for BC and HC participants, of which pentanal, hexanal and decanal levels were mutually lower or higher, which means that their presence in the headspace of VOCs is specific for both blood samples and tissue specimens. This provides rationale for developing diagnostic tests for BC based on altered trace VOCs concentrations using the relatively inexpensive, easy-to-use, portable, and non-invasive E-Nose technology.