BACKGROUND: Fabricating oligonucleotide microarrays with high immobilization efficiency and sensitivity by a simple surface modification route remains highly desirable in the biochip field. Here we develop a facile method to prepare a 3D crosslinking thin hydrogel film on glass slides for high-density immobilization of DNA probes. RESULTS: Thin hydrogel film using poly (ethylene glycol) diacrylate (PEGDA) as crosslinker and glycidyl methacrylate (GMA) as functional monomer was attached on vinyl group-modified glass slides by a photopolymerization technique. The effect of monomer ratios (PEGDA:GMA) on the surface hydrophilicity of the hydrogel layer and the immobilization density of DNA probes were investigated and an optimal monomer ratio of 10:1 and te maximum immobilization density of 1.4 pmol cm -2 were found. Adjusting the hydrophilicity of the hydrogel film effectively suppressed its swelling and produced a typical dense but not porous structure with a flat surface favorable for printing microarrays. The maximum immobilization density and efficiency of oligonucleotide probes on the 3D hydrogel film were 1.4 pmol cm -2 and 83%, respectively, which are superior values to those on a 1D support. The microarray had a wide detection range of target nucleotides from 15.3 pmol L -1 to 62.5 nmol L -1 . In addition, the hybridization assay demonstrated that the microarray showed high sensitivity with a limit of detection of 30.5 pmol L -1 . CONCLUSION:The poly (PEGDA-co-GMA) hydrogel film could be grafted on the glass slide by the photopolymerization technique and the DNA probe was effectively immobilized on the thin hydrogel by reacting with the epoxy groups. This strategy of attaching a thin cross-linked poly(PEGDA-co-GMA) hydrogel layer on glass slides has promising prospects in the fields of bioanalysis and microarrays.