Various histological staining methods have been explored to detect the joint lesions in osteoarthritis (OA), but these histological stains cannot comprehensively present the comparatively complex structures of articular cartilage in knee OA. In addition, no integrated histological staining method can be used to evaluate efficiently both the subzone region and matrix composition in cartilage containing tissues. Therefore, in this study, a novel multichromatic staining method termed TTF staining, using Toluidine Blue (T), Tartrazine (T) and Fast Green (F) sequential combined staining for histological analysis, has been exploited to characterize the changes of matrix components and contents in cartilage during OA and in the bone development. This specific TTF staining profile can be used to differentiate the major compartments of knee joint region, including the synovium, meniscus, multiple subzones of cartilage and subchondral bone. An anterior cruciate ligament transection induced OA model in rat has been established to profoundly present the alterations of glycosaminoglycans in cartilage degeneration by TTF staining profile. The changes of TTF staining profile in the chondrification and ossification centers of the postnatal rat knee joint indicate the developmental features of cartilage matrix during the growth of bone. In summary, we have developed an effective histological staining method that enables us to identify the subzones of cartilage in detail and to define the matrix features of bone development. Therefore, finally using this new TTF staining method may help us to exploit a histopathological grading system to assess cartilage lesions in clinical disease.