A disintegrin and metalloproteinase with thrombospondin motif-4 (ADAMTS-4) plays a pivotal role in degrading aggrecan, which is an early event in cartilage degrading joint diseases such as osteoarthritis (OA). Detection of ADAMTS-4 activity could provide useful clinical information for early diagnosis of such diseases and disease-modifying therapy. Therefore, we developed a ADAMTS-4 detective fluorescent turn-on AuNP probe (ADAMTS-4-D-Au probe) by conjugating gold nanoparticles with a FITC-modified ADAMTS-4-specific peptide (DVQEFRGVTAVIR). When the ADAMTS-4-D-Au probe was incubated with ADAMTS-4, the fluorescence recovered and fluorescence intensity markedly increased in proportion to concentrations of ADAMTS-4 and the probe. A nearly 3-fold increase in fluorescent intensity in response to only 3.9 pM of ADAMTS-4 was detected, whereas almost no fluorescence recovery was observed when the probe was incubated with matrix metalloproteinase (MMP)-1, -3, and -13. These results indicate a relative high sensitivity and specificity of the probe. Moreover, ADAMTS-4-D-Au probe was used to detect ADAMTS-4 activity in synovial fluid from 11 knee surgery patients. A substantial increase in fluorescent intensity was observed in the acute joint injury group as compared to the chronic joint injury and end-stage OA groups, indicating that this simple and low-cost sensing system might serve as a new detection method for ADAMTS-4 activity in biological samples and in screens for inhibitors for ADAMTS-4-related joint diseases. Additionally, this probe could be a potential biomarker for early diagnosis of cartilage-degrading joint diseases.