Nanoplastics (NPs, < 1 μm) have attracted widespread attention due to their potential threats to organisms, ecosystem, and public health. However, there is limited research on the effects of NPs on cephalopods. Sepia esculenta (Mollusks, Cephalopoda, Sepioidea), an economically major species of Sepiidea family in the world, is well received by consumers for its delicious quality and rich nutrition. In the present study, S. esculenta larvae were treated with polystyrene nanoplastics (PS-NPs) at 50 μg/mL for 4 h, and then physiological biomarkers (SOD, GSTs and MDA) and RNA-Seq in the whole body were performed. Research results suggested that PS-NPs exposure had induced oxidative stress of S. esculenta larvae. In addition, several key genes and signaling pathways related to oxidative stress were obtained based on DEGs, GO and KEGG enrichment analyses, as well as PPI construction analysis of transcriptome data. This study enriches the research on the effects of NPs exposure on different organisms and provides insights and references for revealing the mechanisms of oxidative stress caused at the molecular level.