This study aimed to evaluate the effects of capelin roe phospholipid (CPL) on lipid accumulation, fatty acid profile, oxidative stress, and changes in target macromolecular structure in HepG2 cells using multidimensional hyperspectral technique. The results showed that CPL significantly inhibited oleic acid-induced accumulation of intracellular lipids and increased the activity of intracellular antioxidant enzymes (CAT, GSH-Px, SOD). The control and CPL-treated groups promoted accumulation of unsaturated fatty acids and reduced deposition of saturated fatty acids. The therapeutic effects of CPL positively correlated with its concentration. Fourier transform infrared spectroscopy and two-dimensional correlation infrared analysis indicated that CPL decreased the relative contents of intracellular carbohydrate, nucleic acid, –CH2-, and ν(C=O)/νas (CH2+CH3), and maintained the order of intracellular protein structure. Fourier transform infrared spectroscopy could be used for label-free cell sorting. This work provided a direction for the subsequent development of functional lipids in aquaculture.