Extracellular matrix (ECM) is the first line of defense which is an inimitable organelle that perceives biotic and abiotic stresses and reprograms biological processes of host. It also activates innate immune responses in temporal and spatial manner and acts as physical scaffold that prevents the entry of pathogen and microbes in response to stress and hormonal signals. Stem rot, caused by Macrophomina phaseolina adversely affects fiber production in jute. However, how wall related susceptibility affects ECM proteome remains undetermined in bast fiber crops. In order to develop ECM proteome of C. olitorius var. O-4, ECM fraction was isolated by mechanical disruption, CaCl2 extraction and enrichment. ECM fraction purity was assessed by 1-DE profiling in which the ECM fraction was found to be discrete from the crude extract and supernatant recovered during purification steps. Three biological replicates were performed for gel-based jute ECM proteomic analysis. MS/MS analysis of total extract and supernatants were identified as Rubisco or other chloroplastic proteins which were depleted in consecutive steps of purification depicting high degree of ECM purity. Fourteen bands of ECM enriched fraction were identified as canonical ECM localized proteins, e.g., beta-glucosidase, beta-d xylosidase, peroxidase, glycoside hydrolase and peroxiredoxin, confirming jute ECM protein purity and enrichment.