To mimic the structure and functionality of multienzyme complexes, which are widely present in Nature, Pluronic-based micelles were designed to colocalize multiple enzymes. To stabilize the micelles as well as to enable characterization of single enzyme immobilization and multienzyme colocalization by Forster resonance energy transfer (FRET), quantum dots (QDs) were incorporated into the micelles to form Pluronic-QD micelles using a novel microreactor. Model enzymes glucose oxidase (COX) and horseradish peroxidase (HRP) were respectively labeled with fluorescent dyes. The results indicated that FRET occurred between the QDs and dyes that labeled each type of enzyme in single enzyme immobilization studies as well as between the dyes in colocalization studies. These observations were consistent with increases in micelle size after adsorption of dye-enzymes as verified by dynamic light scattering. In addition, the activity of single enzymes was retained after immobilization. An optimized colocalization process improved the overall conversion rate by approximately 100% compared to equivalent concentrations of free enzymes in solution. This study demonstrates a versatile platform for multienzyme colocalization and an effective strategy to characterize multienzyme immobilization and colocalization, which can be applicable to many other multienzyme systems.