Background: Osteosarcoma has a global incidence of 3.4 cases per million annually, with 10–20% of patients presenting with metastasis at diagnosis. Its high metastatic potential is attributed to a highly proliferative cell population and cancer stem cells that drive tumorigenesis and metastasis. This study examines the relationship between CD133 and CXCR4 expression and metastasis in osteosarcoma. Methods: Using a cross-sectional approach, blood serum from osteosarcoma patients diagnosed at two centers was analyzed for CD133 and CXCR4 levels via Reed Biotech ELISA KIT. Absorbance quantified marker levels, and metastasis data were obtained from medical records. A chi-square test assessed the relationship between marker levels and metastasis, with significance set at p < 0.05. Results: Among 40 patients (80% < 40 years), mean CD133 was 0.23 ± 0.02 pg/ml and mean CXCR4 was 6015.82 ± 2345.55 pg/ml. CD133 and CXCR4 levels were significantly associated with metastasis (p = 0.009 and p < 0.001, respectively). Conclusion: These findings suggest that higher expression of CD133 and CXCR4 correlates with increased metastasis in osteosarcoma, underscoring their potential roles in predicting metastatic behavior and aiding in targeted therapeutic strategies. © The Author(s) 2025.