The spread of cancer cells to skeletal tissues which mainly consist of hydroxyapatite (HAp) may indicate some correlation between cancer cells and HAp. It is well accepted that an effective experimental model (in vitro cancer model) which mimics the microenvironment in vivo is of great importance for cancer research. Herein, for the first time, HAp nanoparticles are incorporated into poly(lactic-co-glycolic acid) (PLGA) nanofibers to construct biomimetic 3D nanofibrous scaffolds as an in vitro cancer model. The morphologies, structure, mechanical and thermal properties of the PLGA/HAp scaffolds were characterized. The as-prepared PLGA/HAp scaffolds exhibit decreased fiber diameter, enhanced mechanical properties (31% and 8% improvement in tensile strength and modulus, respectively), and rougher surface over PLGA scaffold. The in vitro biocompatibility of PLGA/HAp scaffolds was evaluated with breast cancer cell line (MCF-7) using PLGA scaffold as control and cell cycle progression was detected by flow cytometry. MTT assay and live staining results demonstrate that PLGA/HAp scaffolds can better support cell viability and proliferation over PLGA scaffold. Analysis of cell cycle reveals that DNA synthesis, cell division, and proliferation of cancer cell are related to the presence of HAp in PLGA nanofibers. Our studies demonstrate the suitability of HAp-incorporated PLGA fibrous scaffolds as a promising platform for 3D cancer cell culture.