The structure changes of α-lactalbumin after binding oleic acid and linoleic acid, including hydrophobic amino acids, hydrophobic regions, tertiary structure, secondary structure, was studied by intrinsic fluorescence, ANS-binding intrinsic fluorescence and circular dichroism spectrum, respectively. The anti-tumor activity of the lactalbumin-oleic acid complex and lactalbumin-linoleic acid complex was measured using the methylene blue method. It can be seen from the fluorescence spectra that a significant red-shift from 331.07 to 337.67 nm and 337.60 nm of α-lactalbumin occurred after binding of oleic acid and linoleic acid, respectively. Together with the ANS-binding spectra, which exhibited a blue-shift (from 516.20 to 514.10 nm and 508.50 nm, respectively) with an increased fluorescence intensity, it can be indicated that binding of oleic acid and linoleic acid lead to the exposure of hydrophobic amino acids and hydrophobic regions. Results of circular dichroism spectra indicated the partial loss of the tertiary structure, and an decrease of β-turn and random coil, which turn to the β-sheet structure. Furthermore, the anti-tumor activity of the two complexes was verified on the three types of tumor cells. This study laid a theoretical basis for the development of anti-tumor drugs.