Extracellular vesicles (EVs) have emerged as promising biomarkers and therapeutic agents, yet their quantification remains technically challenging due to the limitations of conventional methods. Here, a low-cost, fluorescence-based, paper-strip immunoassay is presented for rapid and semi-quantitative estimation of EV concentration, inspired by pH strips. The assay utilizes nitrocellulose membranes functionalized with capture antibodies (anti-CD63, CD9, CD81) and fluorescent dye (ExoBrite™) for EV detection. Systematic optimization of assay parameters—including dye application sequence, incubation time, antibody configuration, and dye concentration—revealed that labeling EVs with dye and incubating on the nitrocellulose paper strips for 20 min yielded the strongest and most reproducible signal. A 200× dilution of ExoBrite™ dye was determined to provide the best balance between sensitivity and specificity. A standard curve generated through twofold serial dilution of EVs from ovarian cancer cell culture medium confirmed a positive, concentration-dependent fluorescence response, establishing a usable dynamic range. Compared to existing technologies, this platform enables fast, simple-to-implement EV quantification using minimal sample volume and equipment. The simplicity and scalability of the method offer strong potential for use in clinical diagnostics and EV research applications.