The detection of nanoscale polymer microspheres in oilfield injection-production fluids is of great significance to the study of water shutoff and profile control mechanism of nanoscale polymer microspheres. However，there is still no report on this aspect. In this paper，a convenient，rapid，and accurate method for the determination of polymer microspheres in oil field injection and production fluids was developed by using high performance liquid chromatography with carefully selecting appropriate detectors，chromatographic columns and mobile phase conditions. Using a double hydroxyl hydrophilic column and 250 mg/L NaH2PO4 aqueous solution as mobile phase，the standard curve of concentration of nanoscale polymer microspheres-peak area of nanoscale polymer microspheres chromatography（retention time at 1.1 min）was drawn. The concentration of nanoscale polymer microspheres could be obtained by substituting the peak area of chromatographic peak at the retention time of 1.1 min into the standard curve. The linear range of the established detection method was 10—2000 mg/L，and the minimum detection quantity was 5 mg/L. The method had very good intraday and daytime precision（RSD<5%）and labeled recovery（>90%），which could be used for the rapid and accurate detection of nanoscle polymer microspheres in the injection and production fluids of actual oil fields.