A localized maskless modification method of polyurethane (PU) films through an atmospheric pressure He/O2 plasma microjet (APPμJ) was proposed. The APPμJ system combines an atmospheric pressure plasma jet (APPJ) with a microfabricated silicon micronozzle with dimension of 30 μm, which has advantages of simple structure and low cost. The possibility of APPμJ in functionalizing PU films with hydroxyl (–OH) groups and covalent grafting of gelatin for improving its biocompatibility was demonstrated. The morphologies and chemical compositions of the modified surface were analyzed by scanning electronic microscopy (SEM), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). The fluorescent images show the modified surface can be divided into four areas with different fluorescence intensity from the center to the outside domain. The distribution of the rings could be controlled by plasma process parameters, such as the treatment time and the flow rate of O2. When the treatment time is 4 to 5 min with the oxygen percentage of 0.6%, the PU film can be effectively local functionalized with the diameter of 170 μm. In addition, the modification mechanism of PU films by the APPμJ is investigated. The localized polymer modified by APPμJ has potential applications in the field of tissue engineering.