This paper proposes a novel technique for fabricating micro patterns of glutaraldehyde (GA)-crosslinked gelatin. It provides another means to crosslink gelatin other than using photo-sensitizing agents, and the micro patterns of GA-crosslinked gelatin can still be made successfully by accessing conventional photolithography. A much less toxic and increased biocompatible approach to strengthening the gelatin microstructures can be developed according to this idea. This paper also describes a potential methodology for using GA-crosslinked gelatin patterns as single-cell culture bases. The best spatial resolution of the micro gelatin bases can reach 10 µm, and the selective growing density of human Mesenchymal stem cells on the gelatin patterns surpasses the density on the glass substrate by 2–3 orders of magnitude.