Raman spectroscopy was used to investigate the order-disorder phase transition of n-eicosane for real-time cooling run. At the phase transition temperature, the integrated intensity of the 1 300 cm-1 spectra range changes greatly, which requires further consideration of 1 300 cm-1 as a reference in the research on polymer and biomembrane using Raman spectroscopy. The experiment support was provided for doubt about order parameter based on 1 130 cm-1 that it did not refer to generally accepted interpretation of an order parameter and it proved that the order definition could just be used as a relative measure method for disorder.