Background: Osteoarthritis is not only a disease of articular cartilage, but also of subchondral bone. It is difficult for ultrasound to evaluate subchondral bone because of permeability. This study aimed to evaluate sequential osteoarthritic changes of articular cartilage and subchondral bone from immobilized knees in rats with a photoacoustic (PA) imaging system. Methods: The unilateral knee joints of adult male rats were immobilized with an internal fixator (IM group) for 4 and 8 weeks. Sham operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). A whole proximal third of the tibia was obtained. The specimens were immersed in normal saline and scanned by the PA imaging system, which can create both ultrasound (US) and PA imaging. After the scanning, specimens were fixed with paraformaldehyde, decalcified and embedded in paraffin. Thinly sliced tissues were prepared for histological evaluation. PA signals from the articular cartilage and subchondral bone were independently evaluated. Results: Gross observations showed that fibrillation occurred in the superficial layers and permeability of the articular cartilage increased. Histological observations showed that the articular cartilage became thicker and the bone marrow space expanded during immobilization. US images by the PA imaging system could evaluate the articular cartilage. PA signals from the articular cartilage and subchondral bone increased with the duration of immobilization and the difference, compared to the control group, was significant. Conclusions: The PA imaging system could evaluate articular cartilage and subchondral bone simultaneously.