A novel microfluidic multiplexed immunohistochemistry (MMIHC) platform was proposed for quantitative pathological diagnosis. Multilayer soft lithography and two-step lithography technologies were used to fabricate MMIHC devices. To overcome the subjective decision of immunohistochemistry (IHC) staining results, image analysis was conducted and quantified with staining intensity and stained area values. By using reversible sealing method between the device and a cellblock sample, not only perfect fluid control for various solutions was exhibited without any leakage, bubble formation and cross-contamination, but also intact sample condition in reaction areas and stable sample preservation were guaranteed. Biomarker examination for estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67 was realized for SK-BR-3 breast cancer cell line at one time. In our experiment, it was revealed that the platform enhanced at least 200-fold reaction efficiency and 24-fold reduction of incubation time. These achievements expect not only savings of time, cost and tissue samples, expanded test for prognostic biomarker candidates and early personalized therapy, but also integration of genomic and proteomic approaches into search for targets in oncology.