The Steptoean Positive Carbon Isotope Excursion (SPICE) event is extensively documented in globally distributed carbonate and organic-rich successions of slope and platform environments during the late Cambrian (ca. 497–494 Ma). Despite numerous studies, its regional manifestation remains controversial because of the major uncertainty of the redox conditions in sedimentary paleoenvironments and its effect on the changes in the biota. Biogeochemical processes that occurred during the SPICE event are important for assessing whether the excursion is associated with the co-evolution of organisms and environments. In this study, we investigated the changes in plankton and paleoenvironment at the molecular level by combining kerogen elemental analysis, catalytic hydropyrolysis (HyPy), and gas chromatography-mass spectrometry (GC-MS) analyses of saturates and aromatics with published carbon isotopic data. The three fluorene series, gammacerane/C 30 hopane (G/C 30 H) and pristine/phytane (Pr/Ph) ratios demonstrated that the shallow shelf seawater changed significantly from relatively reducing to oxidizing and back to reducing conditions during the SPICE interval coupled with a regression-transgression cycle. The phytoplankton during the rise of the SPICE were mainly organic-walled, such as cyanobacteria, various acritarchs, and algae, as evidenced by terpanes, hopanes, and steranes. Significant decreases in phytoplankton diversity and abundance coincided with the peak of the SPICE, corresponding to the most oxidized and hypersaline periods during the SPICE event in the eastern Tarim Basin. Plankton assemblages diversified significantly with increasing living space due to the rise of sea-level during the post-SPICE period. Therefore, this study supports the idea that plankton groups changed dramatically with the environment, and its revolution followed the SPICE into the Great Ordovician Biodiversification Event. • The biological and environmental changes associated with the SPICE event were investigated at the molecular level. • The kerogens were not only sourced from marine plankton but also considerable terrigenous organisms. • The SPICE interval was coupled with a regression-transgression cycle. • The peak of the SPICE corresponded to the lowest sea-level and relatively oxic period in the eastern Tarim Basin. • Plankton changed dramatically with the environments and the SPICE was coincident with the onset of plankton revolution.