Objective: This study aims to investigate proteomic and transcriptomic differences between exosomes derived from sex-specific human embryonic stem cells (hESCs-exo) and human amniotic mesenchymal stem cells (hAMSCs-exo) at the molecular level. Methods: A defined cell culture system optimized for exosome research was established. High-quality exosomes were isolated via ultracentrifugation from male and female lines of hESCs and hAMSCs. Proteomic characterization was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), while miRNA profiles were determined by high-throughput sequencing. Bioinformatics analysis subsequently identified key biological processes and pathways regulated by the exosomes. Results: At both proteomic and transcriptomic levels, exosomes derived from male and female hAMSCs exhibited similar molecular profiles, whereas those originating from male and female hESCs showed significant sex-based differences. Integrated miRNA profiling and proteomic analysis revealed that hESCs-exo were primarily associated with developmental and metabolic processes, while hAMSCs-exo predominantly participated in immune and metabolic functions. Conclusion: Through systematic proteomic and miRNA sequencing analyses of sex-specific exosomes derived from hAMSCs and hESCs, this study predicted their biological functions and revealed their potential applications across multiple domains. These findings provide novel perspectives for selecting optimal cell sources in preclinical and clinical trials.