Several specific tests for cervical screening have been developed recently, including p16/Ki67 dual immunostaining for diagnosing high-risk human papillomavirus positive squamous intraepithelial lesion in the cervix. However, manual screening of cells in an entire glass slide is currently a standard clinical procedure for quantification and interpretation of immunocytochemical features of the cells. Here, we developed a microfluidic device containing an electroactive microwell array with barriers (EMAB) for highly efficient single-cell trapping followed by on-chip immunofluorescence analysis with minimum loss of the sample. EMAB utilizes patterned electrodes at the bottom of cell-sized microwells to trap single cells using dielectrophoresis (DEP) and cell-holding structures behind the microwells to stabilize the position of trapped cells even without DEP. Using the device, we evaluated the performance of p16/Ki67 dual immunostaining of HeLa cells on the chip. The device shows 98% cell-trapping efficiency as well as 92% cell-holding efficiency against the fixed HeLa cells, and we successfully demonstrated high-efficiency on-chip immunofluorescence analysis with minimal loss of sample. p16/Ki67 dual immunostaining using EMAB may be useful for complementary tests for cervical screening in confirming the histopathological diagnosis.