Changes in the levels of lipids are associated with breast cancer (BC). Disease-specific serum free fatty acids (FFAs) were quantified using chip-based direct-infusion nanoelectrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (CBDInanoESI-FTICR MS) in the negative ion mode. Multiple point internal standard calibration curves between the concentration ratios of fatty acids (i.e., C16:1, C18:3, C18:2, C18:1, C20:4, and C22:6) to internal standards (C17:1 for C16:1, C18:3, C18:2, and C18:1, C21:0 for C20:4 and C22:6) and their corresponding intensity ratios were established with a correlation coefficient of greater than 0.986. Data from 342 serum samples including 202 healthy controls and 140 BC patients indicate that serum concentrations of FFAs in patients with BC were significantly decreased compared with those in healthy controls. A panel of C16:1, C18:3, C18:2, C20:4, and C22:6 showed an excellent diagnostic ability to differentiate the patients with early stage BC from healthy controls, with the area under the receiver operating characteristics (ROC) curve of 0.953, a sensitivity of 83.3%, and a specificity of 87.1%. Our findings suggest that these FFAs may be a valuable biomarker panel for the early-stage detection of BC.