Owing to small size，stable property，and good deformation capacity，nanoscale polymer microspheres can migrate into deep formation to enlarge the swept area，thus enhancing waterflooding efficiency. To comprehend the plugging performance of nanoscale polymer microspheres on high-permeability layers，we used the propped fracture evaluation system to simulate the high-permeability channels and injected the solutions of nanoscale polymer microspheres with varying concentrations and particle sizes in different conditions. We analyzed and evaluated the plugging performance of these microspheres on the high-permeability layers according to the change in permeability before and after the injection. As a result，nanoscale polymer microspheres could plug the high-permeability layers. A higher injection rate led to lower plugging performance. As the closing pressure increased，the plugging performance fluctuated. No distinct linear relation was observed between the plugging performance and concentration or particle size of nanoscale polymer microspheres. Instead，concentration and particle size brought about corresponding optimal values of plugging performance. Analysis of dominant factors showed that the injection rate had the greatest influence，followed by closing pressure，and then concentration and particle size of nanoscale polymer microspheres.