Gelatin has been widely used to develop tissue engineering scaffolds because it has many attractive properties. Dendrimer provides a versatile, compositionally and structurally controlled architecture to construct nanomedicine. This study was aimed at developing a novel electrospun dendrimer-gelatin nanofiber scaffold to best mimic natural extracellular matrix (ECM) to promote tissue formation and serve as a reservoir for controlled drug delivery. Starburst™ polyamidoamine (PAMAM) dendrimer G3.5 was covalently bonded to the gelatin backbone and electrospun into nanofibers. Doxycycline (DC), which is an effective antibiotic that has the ability to inhibit matrix metalloproteinase, was encapsulated into the nanofiber scaffold. The electrospun DC-gelatin scaffold provides a bacterial free environment for cell growth and tissue regeneration. The resulting dendrimer-gelatin nanofiber scaffold achieved a unique structural configuration where covalently bound three-dimensional dendritic nanospheres were evenly distributed along the elongated dimension of the nanofiber, and both dendrimer and gelatin had numerous functional groups suitable for accommodating multiple functional entities and high payload of drugs. The development of this new scaffold with the capability of delivering multiple functional entities was an important step towards the use of bioactive nanofibers to facilitate tissue regeneration and controlled drug release.