Three-dimensional (3D) cells in vitro culture are becoming increasingly popular in cancer research because some important signals are lost when cells are cultured in a two-dimensional (2D) substrate. In this work, bacterial cellulose (BC)/gelatin hydrogels were successfully synthesized and were investigated as scaffolds for cancer cells in vitro culture to simulate tumor microenvironment. Their properties and ability to support normal growth of cancer cells were evaluated. In particular, the human breast cancer cell line (MDA-MD-231) was seeded into BC/gelatin scaffolds to investigate their potential in 3D cell in vitro culture. MTT proliferation assay, scanning electron microscopy, hematoxylin and eosin staining and immunofluorescence were used to determine cell proliferation, morphology, adhesion, infiltration, and receptor expression. The in vitro MDA-MD-231 cell culture results demonstrated that cells cultured on the BC/gelatin scaffolds had significant adhesion, proliferation, ingrowth and differentiation. More importantly, MDA-MD-231 cells cultured in BC/gelatin scaffolds retained triple-negative receptor expression, demonstrating that BC/gelatin scaffolds could be used as ideal in vitro culture scaffolds for tumor cells.