Objective: To observe the expression of osteosarcoma stem cell biomarkers Bmi1 and KLF4 in osteosarcoma tissues and explore their value in the diagnosis, treatment and prognosis of osteosarcoma. Methods Using retrospective research methods, 51 patients (28 males and 23 females) with osteosarcoma who were surgically resected and diagnosed by pathology in the Second Hospital of Shanxi Medical University from October 2009 to July 2019 were used as the experimental group. The age of cases varies from 10 to 67 (average 27.04) years old, and bone tissues adjacent to the tumor were taken from 10 samples as the normal control group. Immunohistochemical method was used to assess the expression levels of Bmi1 and KLF4 in 51 patients with osteosarcoma and 10 cases of paraneoplastic bone tissue specimens. Chi-square test was applied to analyze the relationship between the expression of Bmi1 and KLF4 and the clinical pathological data of patients. Correlation analysis was analyzed by the number of connections. The survival rate of patients was calculated by the Kaplan-Meier method. The log rank univariate analysis and Cox regression multivariate analysis were carried out to evaluate the prognostic value. Results The positive expression rates of Bmi1 and KLF4 in the osteosarcoma group were 78.43% (40/51) and 80.39% (41/51), respectively, and in the bone tissue group were both 3/10. The difference of the positive expression rates of Bmi1 and KLF4 in the osteosarcoma group and bone tissue group was statistically significant (P < 0.05). In osteosarcoma group, the expression levels of Bmi1 and KLF4 were positively correlated (R = 0.399, P < 0.01). Bmi1 protein-positive, KLF4 protein-positive, and Bmi1 and KLF4 protein double-positive expression were statistically significant in Enneking surgical staging, lung metastasis, and pathological typing (all P < 0.05), but there was no statistically significant difference between different ages, genders, local recurrences, and tumor sizes (all P > 0.05). Conclusion The expression of Bmi1 and KLF4 in osteosarcoma tissue was significantly higher than that in surrounding bone tissues, and their positive expression is a risk factor for the prognosis of patients with osteosarcoma.