SH-SY5Y cell line treated with 6-hydroxydopamine (6-OHDA) is a classical Parkinson’s disease (PD). In the present study, synchrotron-based Fourier transform infrared (FTIR) microspectroscopy was used to analyze the biochemical composition of SH-SY5Y cell line treated with 6-OHDA. The detailed spectral analyses show the significant changes in cellular compositions such as lipids, proteins and nucleic acids in SH-SY5Y cells treated with 6-OHDA compared to control SH-SY5Y cells. As a result, the unsaturation levels of phospholipids decrease in SH-SY5Y cells treated with 6-OHDA compared to control cells, the analysis of protein secondary structure shows the significantly higher ratio of β-sheet in PD cells compared to that of control cells, and the content of nuclear acid is highly decreased compared to that of control cells, suggesting that 6-OHDA induces the serious oxidative damage in SH-SY5Y cells. These findings suggest that SR-FTIR is an effective and precise technical tool to probe the biochemical changes of cells and then evaluate the pathological damage in cells.