Background: To systematically analyze the components of excretory-secretory protein (ESP) of Spirometra mansoni. Methods: The adult and plerocercoid of S. mansoni were cultivated in vitro. Then, the differentially expressed proteins (DEPs) were enriched and protein components were examined using Data Independent Acquisition (DIA) mode. Furthermore, bioinformatics analysis was performed on DEPs. Results: A total of 944 proteins were identified, including 580 plerocercoid-specific proteins, while no specific proteins were found in adult. Quantitative analysis showed that 607 proteins exhibited significant differential expression, with 390 proteins upregulated in the plerocercoid and 217 upregulated in the adult. The Gene Ontology functional annotation showed that the upregulated proteins in the plerocercoid were significantly enriched in nitrogen compound metabolism, proteasome core complexes, and ion binding. Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed the DEPs were highly correlated with signal transduction, signal transportation, and catabolism pathways. Meanwhile, metabolic network analysis showed that key pathways included the pentose phosphate pathway and glycolysis/gluconeogenesis. The protein-protein interaction network further revealed that RL40 played a pivotal role in functional regulatory nodes. In addition, indirect ELISA showed that ES immunization induced a Th1/Th2 mixed immune response in mice, dominated by Th1. Cytokine detection further verified that ES had good immunogenicity, and could activate both humoral and cellular immune responses. Conclusions: This study reveals the differential expression profile of ESPs between the adult and plerocercoid of S. mansoni for the first time. Findings in this study may demonstrate that ESP in the plerocercoid can induce a Th1-biased Th1/Th2 mixed immune response, offering potential reference for the diagnosis and prevention of sparganosis.