One of the major limitations of adenovirus as an efficient gene delivery vehicle is its transient nature of transgene expression. At present, no biodegradable release system exists that permits the sustained release of gene delivery. This study aimed to optimize a biodegradable fibrin scaffold for sustained adenoviral mediated gene delivery. Scaffold formulations were fabricated incorporating an adenoviral vector encoding beta-galactosidase, and each scaffold was incubated in elution fluid. The level of transfection of cells was measured to assess virus diffusion from each scaffold at different time periods. Results showed that viral particles were present in the elution fluid and had the ability to transfect cells at different time points. Depending on the scaffold formulation, variable rates of diffusion were seen. Analysis of scaffold microarchitecture using stereo pairs showed that variable pore depth is seen in different fibrin scaffold concentration, which may be a determining factor in the diffusion rate. It was concluded that sustained release of the adenovirus could be attained up to 192 h at the optimal concentration of 60 mg/mL fibrinogen and 4 IU thrombin.