Bacterial Extracellular vesicles or BEVs are infinitesimal bi-layered lipid vesicles secreted from non-pathogenic and pathogenic bacteria to be used as a targeted drug-delivering system (DDS). Our study compared the biophysical and molecular characteristics of OMVs isolated from E. coli BL21 (DE3) transformed with the plasmid pET28a-His6-Signal Anchoring Green Fluorescent Protein (SAGFP) grown in Luria-Bertani and M9 minimal media to select the best medium for the growth of engineered bacterial OMVs. Isolation of BEVs using the ultracentrifugation method yielded bacterial vesicles with smaller mean sizes and excellent recovery of vesicle morphology. The evaluation of the size distribution profile of BEVs using the Dynamic light-scattering technique assessed for vesicle size with particle aggregation ranging from 20-750 nm. 3D interactive surface plots and particle size-distribution analyses of BEVs obtained from their HR-TEM results depicted slight morphological dissimilarities. Investigation on SAGFP cargo sort into vesicles using fluorescence spectrophotometry, and fluorescent microscopy solidates the expression of SAGFP in OMVs isolated from transformed bacterial culture when the expression was induced with 10 mM Isopropyl-D-1-thiogalactopyranoside (IPTG) at OD₆₀₀ = 0.6 in both media. Isolation of engineered BEVs (eBEVs) grown in LB media had higher vesicle yield and good particle recovery that could be directly incorporated into targeted therapeutics. However, BEVs grown in minimal media had good particle purity with increased protein concentration but yielded vesicles with lower particle recovery. This comparative investigation should help analyze the efficacy and characteristics of engineered BEVs grown in two different media and provide a robust and straightforward method to engineer BEVs. These engineered BEVs could be utilized as both fluorescent probes and a drug-delivering vehicle in targeted therapeutics.