The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance ( < 0.05) in nine elbows. Correlation analyses proved statistical significance ( < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant ( < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance ( < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance ( < 0.05) between day 14 and the control group.