Background: Osteosarcoma (OS) is one of the most common malignant bone tumors and specific microRNAs (miRNAs) are closely associated with malignant OS progression. In this study, we examined the role of microRNA-193b-3p (miR-193b) and the involvement of autophagy and apoptosis in the chemosensitivity of OS cells. Methods: We employed qRT-PCR, Western blot, and immunohistochemistry to examine the expression levels of miR-193b, flap endonuclease 1 (FEN1), and autophagy-related proteins. Apoptosis was determined by flow cytometry using an Annexin V-FITC/PI apoptosis detection kit. Luciferase reporter assays confirmed the relationship between miR-193b and FEN1. Results: miR-193b was downregulated in OS compared to adjacent normal tissues (p < 0.05). miR-193b overexpression in the OS cell lines induced autophagy and apoptosis, as shown by Western blotting and flow cytometry. Knockdown of FEN1, a structure-specific nuclease overexpressed in OS tissues (p < 0.001), induced apoptosis through activation of autophagy. Luciferase reporter assays confirmed that FEN1 is a direct target of miR-193b, FEN1 knockdown reinforced miR-193b induced apoptosis. Moreover, miR-193b expression enhanced epirubicin-induced autophagy and apoptosis. Conclusion: Collectively, the results showed that miR-193b/FEN1 may serve as a novel therapeutic target for OS aimed mainly at the induction of autophagy and apoptosis. The miR-193b/FEN1 axis increased the chemosensitivity of OS cells, while activation of autophagy enhanced the anticancer effects of epirubicin.