Objective: To construct a gene signature centered on the STAT3/P53 signaling pathway and clarify its clinical value in prognosis assessment for osteosarcoma patients through direct comparison between the control group and the observation group, providing a reference for personalized treatment. Methods: Eighty osteosarcoma patients admitted to the orthopedics department of our hospital from May 2024 to October 2025 were selected as the study subjects. The expression levels of 12 core genes (CDKN1A, BCL2, MDM2, etc.) in the STAT3/P53 pathway in tumor tissues were detected using real-time fluorescent quantitative PCR (qRT-PCR). Using the median of the gene signature risk score as the cutoff, patients were divided into a high-risk group (observation group, n = 40) and a low-risk group (control group, n = 40). Clinical and pathological characteristics, core gene expression patterns, treatment responses, and short-term prognosis outcomes were compared between the two groups. Results: There were no statistically significant differences between the observation group and the control group in terms of age, gender, and tumor location (all P > 0.05). However, significant differences were observed in the maximum tumor diameter, Enneking stage, and LDH level (all P < 0.001). The expression levels of oncogenes in the observation group were significantly higher than those in the control group, while the expression levels of tumor suppressor genes were significantly lower. The chemotherapy response rate in the observation group was significantly lower than that in the control group (χ² = 12.170, P = 0.001). After 3 months of follow-up, the recurrence and metastasis rate in the observation group was significantly higher than that in the control group, with a statistically significant difference (χ² = 8.658, P = 0.003). Conclusion: The gene signature based on the STAT3/P53 pathway can effectively distinguish between high-risk and low-risk osteosarcoma patients, with significant differences observed between the two groups in terms of clinical characteristics, gene expression, and short-term prognosis. This gene signature provides a reliable basis for prognostic prediction and the formulation of treatment plans.