Functional microbeads have been widely applied in molecular identification and other biochemical applications in the past decade, owing to the compatibility with flow cytometry and the commercially available microbeads for a wide range of molecular identification. Nevertheless, there is still a technical hurdle caused by the significant sample volume required (∼50 μl), limited molecular detection limit (∼20 pg/ml), complicated liquid/microbead handling procedures, and the long reaction time (>2 h). In this work, we optimize the operation of an automated microbead-based microfluidic device for the reagent mixing and the dynamic cytokine detection. In particular, we adopt fluorescence microscopy for quantification of multiple microbeads in each microchamber instead of flow cytometry for a lower detection limit. The operation parameters are then configured for improved measurement performance. As demonstrated, we consider the cytokine secretion of human macrophage-differentiating lymphocytes stimulated by lipopolysaccharides. We examine requirements on the mixing duration, minimal sample volume, and the image analysis scheme for the smaller biosample volume (<5 μl), the lower cytokine detection limit (∼5 pg/ml), and shorter process time (∼30 min). Importantly, this microfluidic strategy can be further extended in the molecular profiling using other functional microbeads for a broad range of biomedical applications.