SignificanceInspired by the widely present multienzyme complexes in nature that enable highly cooperative catalytic mechanisms, we designed a biomimetic dual‐functionalized nanoparticle‐based platform for colocalizing multiple enzymes. The use of nanoscale materials together with a novel sequential colocalization approach with two model enzymes [glucose oxidase (GOX) and horseradish peroxidase] resulted in a 100% increase in the overall conversion rate compared to the equivalent amount of free enzymes in solution and a physical mixture of individual immobilized enzymes on nanoparticles. GOX is an important enzyme used in glucose biosensors for diagnostics. Colocalizing GOX with peroxidase allows for colorimetric visualization of the peroxide formed that enables monitoring glucose levels in solution. This platform can be readily applicable to other multienzyme systems as well. © 2012 American Institute of Chemical Engineers AIChE J, 59: 355–360, 2013