The objective of this work is generation of propolis/polyvinyl alcohol (PVA) scaffold by electrospinning for 3D cell culture. Here, PVA used as co-spinning agent since propolis alone cannot be easily processed by electrospinning methodology. Propolis takes charge in maximizing biological aspect of scaffold to facilitate cell attachment and proliferation. Morphological analysis showed size of the electrospun nanofibers varied between 172-523 nm and 345-687 nm in diameter, for non-crosslinked and crosslinked scaffolds, respectively. Incorporation of propolis resulted in desired surface properties of hybrid matrix, where hybrid scaffolds highly favored protein adsorption. To examine cell compatibility, NIH-3T3 and HeLa cells were seeded on propolis/PVA hybrid scaffold. Results confirmed that integration of propolis supported cell adhesion and cell proliferation. Also, results indicated electrospun propolis/PVA hybrid scaffold provide suitable microenvironment for cell culturing. Therefore, developed hybrid scaffold could be considered as potential candidate for 3D cell culture and tissue engineering.