This paper reports the fabrication of electrospun polydimethylsiloxane (PDMS) membranes/scaffolds that are suitable for three-dimensional (3D) cell culture. Through modification the ratio between PDMS and polymethylmethacrylate (PMMA) as carrier polymer, we report the possibility of increasing PDMS weight ratio of up to 6 for electrospinning. Increasing the PDMS content increases the fiber diameter, the pore size, and the hydrophobicity. To our best knowledge, this is the first report describing beads-free, durable and portable electrospun membrane with maximum content of PDMS suitable for cell culture applications. To show the proof-of-concept, we successfully cultured epithelial lung cancer cells on these membranes in a static well plate without surface modification. Surprisingly, due to three-dimensional (3D) and hydrophobic nature of the electrospun fibers, cells aggregated into 3D multicellular spheroids. These easily detachable and cost-effective scaffolds with controllable thicknesses and high tensile strength are good candidates for cell-stretching devices, organ-on-a-chip devices, tissue engineering and studies of non-adherent mammalian cancer stem cells.