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The two‐dimensional (2D) models of breast cancer still exhibit a limited success. Whereas, three‐dimensional (3D) models provide more similar conditions to the tumor for growth of cancer cells. In this regard, a 3D in vivo model of breast cancer using 4 T1 cells and chitosan‐based thermosensitive hydrogel were designed. Chitosan/β‐glycerol phosphate hydrogel (Ch/β‐GP) was prepared with a final ratio of 2% and 10%. The hydrogel properties were examined by Fourier transformed infrared spectroscopy, MTT assay, pH, scanning electron microscopy, and biodegradability assay. 3D model of breast cancer was induced by injection of 1 × 10⁶ 4 T1 cells in 100 μl hydrogel and 2D model by injection of 1 × 10⁶ 4 T1 cells in 100 μl phosphate‐buffered saline (PBS) subcutaneously. After 3 weeks, induced tumors were evaluated by size and weight determination, ultrasound, hematoxylin‐ and eosin and …