We successfully demonstrate the in situ formation by X-ray irradiation induced polymerization of a three-dimensional (3D) hydrogel based on poly(ethylene glycol) (PEG) diacrylate (PEG DA), with the option of heparin incorporation to enhance cell adhesion. The polymerization reaction can be completed in tens of seconds, thanks to the fast and uniform X-ray induced process. Eliminating polymerization initiators and the residual unreacted monomers improves the biocompatibility and stability of the hydrogel. The photopolymerization mechanism can be locally controlled by using a trimmed or a focused X-ray beam to allow precise in vivo administration deep in the tissue, without surgery. We also demonstrate that photopolymerized hydrogels containing polyethylenimine (PEI)/nucleic acid (DNA or small interfering RNA, siRNA) nanoparticles (NPs) can deliver such nanoparticles. The hydrogel is biocompatible with mice and is not toxic to epithelial cells.