Nanofibre structures of self-assembling peptides as biological materials have recently been studied for their promising uses in tissue engineering and biomedical research. We report here a self-assembling peptide RADA16 that forms nanofibre scaffold to be used as a three-dimensional (3D) cell culture matrix. The 16-residue peptide forms a P-sheet structure and undergoes molecular self-assembly into nanofibres and eventually into a higher-order scaffold hydrogel. In this study, we indicate that the nanofibres assemble into much stiffer scaffold networks which can mimic the tumor microenvironment in vivo more effectively. Circular dichroism spectroscopy, atomic force microscopy, transmission electron microscopy, and rheology were used to study the structure of the peptide nanofibres and the kinetics of the cell culture scaffold formation. The change of this assembly into stiffer scaffold is important for a 3D cancer cell culture. We have studied cellular behaviour of human lung cancer cell A549 within RADA16 nanofibre scaffold including morphology, growth, drug resistance, and adhesion ability to elucidate its application in cancer cell research. Different cell morphologies in a 3D scaffold and on two-dimensional (2D) plastic may be related to the enhanced drug resistance of A549 cell colonies in a 3D culture which forms a 3D gradient to mimic the in vivo conditions and modifies the cellular behaviour that are missing in 2D monolayer culture. Adhesion assay also shows that the 3D cell colonies have significantly different adhesion abilities to ECM proteins of laminin and collagen IV due to the variations of cell-cell and cell-matrix interactions, compared to the 2D cells. Our data suggest that self-assembling peptide RADA16 nanofibre scaffold could provide new insights into cancer studies.