Abstract Circulating extracellular vesicles (EVs) contain molecular footprints from their cell of origin and may provide potential non-invasive access for detection, characterization, and monitoring of numerous diseases. Despite their growing promise, the integrated proteo-transcriptomic landscape of EVs and their donor cells remain poorly understood. To assess their cargo, we conducted small RNA sequencing and mass spectrometry (LC-MS/MS) of EVs isolated from in vitro cancer cell culture and prostate cancer patients’ serum. Here, we report that EVs enrich for distinct molecular cargo, and their proteo-transcriptome is predominantly different from their cancer cell of origin, implicating a coordinated disposal and delivery mechanism. We have discovered that EVs package their cargo in a non-random fusion, as their most enriched RNAs and proteins are not the most abundant cargo from their donor cells. We show that EVs enrich for 4 times more cytoskeletal and 2 times extracellular proteins than their donor cells. While the donor cells carry 10 times more mitochondrial and 3 times nuclear proteins than their EVs. EVs predominantly (40-60%) enrich for small RNA (~15-200 nucleotides) molecules that implicate cell differentiation, development, and signaling signatures. Finally, our integrated proteo-transcriptomic analyses reveal that EVs are enriched of RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, SRSF1/2), implicating an interrelated mechanism that may protect and regulate transcripts until a biological function is achieved. Based on these results, we envision that the next-generation clinical assays will take an integrative multi-omic (proteomic and transcriptomic) approach for liquid biopsy in numerous diseases.