The objective of this work is to demonstrate the feasibility of fabrication of cross-linked polyethyleneimine microfibers by a reactive photo-electrospinning technology. Linear polyethyleneimine (L-PEI) has been grafted with cross-linkable methacrylate moiety by reaction with glycidyl methacrylate (GMA), enabling the polymer to cross-link upon UV exposure. The photo-cross-linking reaction was characterized by a photo-rheometer. Neat L-PEI or methacrylated L-PEI tends to aggregate rendering it very difficult to electrospin into microfibers. A high molecular weight polyvinylpyrrolidone (PVP) is an efficient chain entanglement enhancer for both L-PEI and methacrylated L-PEI and helpful to maintain fibrous structure. An optimized composition consisted of 10% methacrylated L-PEI (less than 14.8% methacrylation of total L-PEI) combined with 2% PVP and 1% photoinitiator in ethanol was successfully electrospun into smooth cross-linked microfibers using the reactive electrospinning device. Diameters of cross-linked fibers can be controlled from 419 nm to 2 mu m depending on methacrylation degree and UV irradiation intensity. The resultant cross-linked L-PEI microfibers have demonstrated significantly improved solvent resistance, thermal stability, and mechanical properties. The distinguished characteristics of this novel reactive electrospinning technology are the high cross-linking efficiency and minimal toxic chemical residues in the products. The stability of the fibers can be readily modified and controlled by the cross-linking degree, which is of great importance for biomedical applications.