Early detection and diagnosis of knee osteoarthritis (OA) in football players and preventive measures to delay cartilage degeneration are crucial to prolong football players' careers and to reduce the waste of coaching and medical resources. In this study, we intended to use the immunochromatographic urine strip together with the International Knee Documentation Committee (IKDC) subjective knee evaluation form and the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index to assess the therapeutic efficacy of intra-articular injections of hyaluronic acid (HA) on knee OA in football players. The 18 football players showed lower IKDC and WOMAC scores compared to the 42 healthy subjects. The players demonstrated significantly improved IKDC scores and WOMAC scores, signifying that the improvement of knee function after the injections of HA. Importantly, 70% of the player's improvements of OA can be detected and reflected from this urine strip. The results revealed that combined the immunochromatographic urine strip with the IKDC and WOMAC could be used to assess the therapeutic efficacy of HA injections on OA.