Poly (ethylene imine) (PEI) is a polycation widely used for DNA transfection to cells but also applied as primary polycation for layer-by-layer (LBL) assembly of polyelectrolytes. The aim of the present study was to investigate the effect of modification with PEI on the biocompatibility of poly (l-lactide) (PLLA) films. PEI with different molecular weight was immobilized on PLLA by either adsorption or covalent binding. Cell morphologies, immuno-fluorescence staining, cell proliferation by lactate dehydrogenase assay and cell differentiation by alkaline phosphatase assay were utilized to assess the biocompatibility of the modified PLLA using osteoblast cell line MG63. Results revealed that PEI modification remarkably improved cell adhesion, viability, proliferation and function compared with plain PLLA. Hence, PEI-modified PLLA is acceptable as transfection vehicle for engineering of bone and other tissues, or as primary layer to allow LBL assembly to generate biomimetic surface coatings.