“Plastisphere”, microbial communities colonizing plastic debris, has sparked global concern for marine ecosystems. Microbiome inhabiting this novel human-made niche has been increasingly characterized; however, whether the plastisphere holds crucial roles in biogeochemical cycling remains largely unknown. Here we evaluate the potential of plastisphere in biotic and abiotic denitrification and nitrous oxide (N₂O) production in estuaries. Biofilm formation provides anoxic conditions favoring denitrifiers. Comparing with surrounding bulk water, plastisphere exhibits a higher denitrifying activity and N₂O production, suggesting an overlooked N₂O source. Regardless of plastisphere and bulk water, bacterial and fungal denitrifications are the main regulators for N₂O production instead of chemodenitrification. However, the contributions of bacteria and fungi in the plastisphere are different from those in bulk water, indicating a distinct N₂O production pattern in the plastisphere. These findings pinpoint plastisphere as a N₂O source, and provide insights into roles of the new biotope in biogeochemical cycling in the Anthropocene.