Objective To detect the expression of phosphatidylinositol 3-kinase (PIK3CA) and glutathione-S transferase-π (GST-π) in osteosarcoma tissues and to explore its effect on the multidrug resistance of osteosarcoma. Methods Thirty-three cases of pathological tissue specimens of osteosarcoma from Renmin Hospital of Wuhan University from January 2009 to June 2017 were collected. The expression of PIK3CA and GST-π of 33 cases of osteosarcoma specimens was detected by immunohistochemistry (Envision method) and Western blot method. The differences between the chemotherapy-resistant group (7 cases) and the chemotherapy-effective group (26 cases) were compared. The results were analyzed combined with clinicopathological factors. Results The average optical density of PIK3CA in the chemotherapy-resistant group and chemotherapy-effective group was (11.089±2.147) and (6.938±2.065) respectively, and there was a significant difference between the two groups (P < 0.05); the average optical density of GST-π in the chemotherapy-resistant group and chemotherapy-effective group was (15.871±2.678) and (7.693±2.443), and there was a significant difference between the two groups (P < 0.05). The average optical density of PIK3CA and GST-π by Western blot method had significant differences between the two groups (P < 0.05). The expression of PIK3CA and GST-π in osteosarcoma was correlated (r = 0.68, P < 0.01). Conclusion PIK3CA and GST-π are over expressed in osteosarcoma chemotherapy-resistant group, and the expression of PIK3CA and GST-π is positively correlated with each other, which may be one of the mechanisms of drug resistance in osteosarcoma.