Antarctic palaeoclimate evolution and vegetation history after the formation of a continent-scale cryosphere at the Eocene–Oligocene boundary, 33.9 million years ago, has remained a matter of controversy. In particular, the reconstruction of terrestrial climate and vegetation has been strongly hampered by uncertainties in unambiguously identifying non-reworked as opposed to reworked sporomorphs that have been transported into Antarctic marine sedimentary records by waxing and waning ice sheets. Whereas reworked sporomorph grains over longer non-successive geological timescales are easily identifiable within younger sporomorph assemblages (e.g. Permian sporomorphs in Pliocene sediments), distinguishing non-reworked from reworked material in palynological assemblages over successive geological time periods (e.g. Eocene sporomorphs in Oligocene sediments) has remained problematic. This study presents a new quantitative approach to identifying non-reworked pollen assemblages in marine sediment cores from circum-Antarctic waters. We measured the fluorescence colour signature, including red, green, and blue fluorescence; brightness; intensity; and saturation values of selected pollen and spore taxa from Eocene, Oligocene, and Miocene sediments from the Wilkes Land margin Site U1356 (East Antarctica) recovered during Integrated Ocean Drilling Program (IODP) Expedition 318. Our study identified statistically significant differences in red-fluorescence values of non-reworked sporomorph taxa against age. We conclude that red fluorescence is a reliable parameter for identifying the presence of non-reworked pollen and spores in Antarctic marine sediment records from the circum-Antarctic realm that are influenced by glaciation and extensive reworking. Our study provides a new tool to accurately reconstruct Cenozoic terrestrial climate change on Antarctica using fossil pollen and spores.