Bacterial cellulose (BC) has emerged as a biomaterial for diverse biomedical applications owing to its unique structural, physico-chemical, mechanical, and biological features. Its porous geometry and three-dimensional fibrous structure allow the impregnation of various materials into its matrix. The current study was aimed to fabricate 3D scaffolds of bacterial cellulose and chitosan (BC-Chi) through a one-step ex situ solution impregnation strategy and analyze the scaffold interaction with the ovarian cancer cell lines (A2780). Field emission scanning electron microscopy (FE-SEM) showed successful impregnation of chitosan into the BC matrix. Phase-contrast and confocal microscopy analyses revealed that human ovarian cancer cell lines (A2780) were adhered not only to the surface but deeply infiltrated into the matrix of BC-Chi scaffold. WST-1 assay, histology analysis, and cytoskeleton and nuclear staining showed high viability, proliferation, and infiltration of A2780 cell lines into the scaffold. The RT-PCR analysis revealed a decreased mRNA level of Notch receptors, indicating a strong cell-scaffold interaction. The improved biocompatibility, non-toxicity, and 3D structure of fabricated BC-Chi scaffold justify its potential applications diagnosis of ovarian cancer in vivo.