Selenium (Se) is an essential element for mammals, and its deficiency in the diet is a global problem. Agronomic biofortification through exogenous Se provides a valuable strategy to enhance human Se intake. Selenium nanoparticles (SeNPs) have been regarded to be higher bioavailability and less toxicity in comparison with selenite and selenate. Still, little has been known about the mechanism of their metabolism in plants. Soybean (Glycine max L.) can enrich Se, providing an ideal carrier for Se biofortification. In this study, soybean sprouts were treated with SeNPs, and a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing was applied to clarify the underlying molecular mechanism of SeNPs metabolism. A total of 74,662 nonredundant transcripts were obtained, and 2109 transcription factors, 9687 alternative splice events, and 3309 long non-coding RNAs (lncRNAs) were predicted, respectively. KEGG enrichment analysis of the DEGs revealed that metabolic pathways, biosynthesis of secondary metabolites, and peroxisome were most enriched both in roots and leaves after exposure to SeNPs. A total of 117 transcripts were identified to be putatively involved in SeNPs transport and biotransformation in soybean. The top six hub genes and their closely coexpressed Se metabolism-related genes, such as adenylylsulfate reductase (APR3), methionine-tRNA ligase (SYM), and chloroplastic Nifs-like cysteine desulfurases (CNIF1), were screened by WGCNA and identified to play crucial roles in SeNPs accumulation and tolerance in soybean. Finally, a putative metabolism pathway of SeNPs in soybean was proposed. These findings have provided a theoretical foundation for future elucidation of the mechanism of SeNPs metabolism in plants.