Duchenne muscular dystrophy (DMD) is a muscular disorder, affecting up to 1 in 3500 births worldwide. The disease is caused by the almost complete absence of dystrophin protein due to out-of-frame mutations in the DMD gene. The loss of dystrophin is characterized by progressive muscle weakness and wasting, eventually causing skeletal and cardiac muscle degeneration, respiratory and cardiac failure, and death before the age of 30. Currently, there are no effective cures available for DMD; therefore, therapy is limited to management of symptoms, including physiotherapy and steroids medication. Antisense oligonucleotides (AONs) exon skipping is one of the most promising therapeutic strategies for DMD, designed to skip a specific DMD exon, which produces a shortened transcript but a functional dystrophin protein. The use of carriers can be an efficient approach for AON delivery, particularly in order to increase AONs stability and to enhance cellular uptake. Here we designed a biomaterial strategy for encapsulation of therapeutic AONs in hydrogels as a part of a controlled release system that can improve the pharmacokinetic properties of the AONs. Specifically, we used PEG-fibrinogen (PF) hydrogel-based microspheres for the delivery of the AONs in the treatment of DMD. The hypothesis was that this microsphere device would greatly decrease the overall administered dosage, thus reducing any adverse effects associated with their administration. The specific AONs used were intended to facilitate dystrophin expression and the device was used to achieve controlled administration of the AONs in vivo. We investigated the effects of increased cross-linking density of the hydrogel on AONs encapsulation and the release rate from the hydrogel. The resulting PEG-fibrinogen microspheres were spherical, with a diameter of approximately 90 µm and with a biphasic release profile of AONs modified by the cross-linking density of the hydrogel. Cellular uptake and localization of AONs in mouse C2C12 muscle cells demonstrated that AONs released from the microspheres well penetrated into the cells. The results indicate that this delivery system could represent a potential therapy for DMD.