Sepsis is a leading cause of death worldwide. In this work, a multiparameter affinity microchip was developed for faster sepsis diagnosis, which can reduce the mortality caused by late validation. The separation device captured cells expressing CD25, CD64, and CD69 into discrete antibody regions. The performance of multiparameter cell separation microchips was compared with flow cytometry analysis and validated with samples of septic patients (n = 15) and healthy volunteers (n = 10). The total analysis time was 2 h. Results showed that total on-chip cell counts for both CD64 and CD69 regions were linear with antigen expression levels. The difference between cell capture for septic and healthy samples was statistically significant (CD64: p = 0.0033; CD69: p = 0.0221, 95% confidence interval), indicating that sepsis is distinguishable based on microfluidic cell capture. For on-chip detection of CD64+ and CD69+ leukocytes, the AUC was 0.95 and 0.78, respectively. The combination of CD64 and CD69 for sepsis diagnosis had the AUC of 0.98, indicating the improved and excellent diagnostic performance of multiple parameters.