Abstract Background Emerging evidence suggests that extracellular vesicles (EVs) can play roles in inflammatory processes and joint degradation in primary osteoarthritis (OA), a common age-associated joint disease. EV subpopulations express tetraspanins and platelet markers that may reflect OA pathogenesis. The present study investigated the associations between these EV surface markers and articular cartilage degradation, subjectively and objectively assessed pain, and functional limitations in primary knee OA (KOA). Methods Serum EVs were determined by high-sensitivity flow cytometry (large CD61⁺ EVs) and single particle interferometric reflectance imaging sensor (small CD41⁺, CD63⁺, CD81⁺, and CD9⁺ EVs) from end-stage KOA patients and controls (n = 8 per group). Knee pain and physical functions were assessed with several health- and pain-related questionnaires, established measurements of physical medicine, and neuromuscular examination. The obtained data were analyzed using supervised and unsupervised univariate and multivariate models. Results With the combined dataset of cartilage thickness, knee function, pain, sensation, and EV molecular signatures, we identified highly correlated groups of variables and found several EV markers that were statistically significant predictors of pain, physical limitations, and other aspects of well-being for KOA patients, for instance CD41⁺/CD63⁺/CD9⁺ small EVs associated with the range of motion of the knee, physical performance, and pain sensitivity. Conclusions Particular serum EV subpopulations showed clear associations with KOA pain and functional limitations, suggesting that their implications in OA pathophysiology warrant further study.