An innovative reflective near-infrared (NIR) fiber probe with flexibility and convenience was designed for biomedical in-situ detection. When the brand-new fiber probe structure and fiber arrangement were designed, further, a gradient-index (grin) lens was coupled to the top of the reflective NIR fiber bundles to make it have higher measurement accuracy and collection efficiency. When measuring NIR spectra of sucrose samples by coupling the grin NIR fiber probe to Fourier transform near infrared spectrometer, it was found that the grin NIR fiber probe had the advantages of convenience, high efficiency and spectral repeatability and SNR. The NIR spectra of articular cartilage at the femoral end of the canine knee joint were measured in situ by the grin NIR fiber probe and were preprocessed by first-derivative quadratic polynomials 21-point Savitzky-Golay smoothing for principal component analysis and Fisher discrimination analysis (PCA-FDA). The correct recognition accuracies of PCA-FDA model in initial cases and cross validation cases were 97.62% and 90.47%, respectively, as well as 96.43% for the prediction set, which confirms the effectiveness of NIR fiber probe detection in situ and the feasibility of osteoarthritis recognition and lays the foundation for basic research and early clinical diagnosis of osteoarthritis.