Chemiluminescence (CL) functionalized materials have found tremendous value in developing CL assays for clinical assays and point-of-care tests. To date, the design and optimization of these materials have mainly relied on conventional trial-and-error procedures in which the ensemble performance is evaluated using conditional experiments. Here we have built an optical microscope to acquire the CL emission from single magnetic-polymer hybrid microbeads functionalized with luminol analogues, and to access the CL kinetics of each individual particle. It was incidentally found that a minor subpopulation of microbeads exhibited intense and delayed CL emission while the majority showed transient and weak emission. Structural characterization of the very same individual particles uncovered that the amorphous multi-core microstructures were responsible for the enhanced encapsulation efficiency and optimized CL reaction kinetics. Guided by this knowledge stemming from single particle CL imaging, the synthesis procedure was rationally optimized to enrich the portion of microbeads with better CL performance, which was validated by both single particle imaging and the significantly improved analytical performance at the ensemble level. The present work not only demonstrates the CL imaging and CL kinetics curve of single microbeads for the first time, but also sets a clear example showing the capability of single particle studies to investigate the structure-activity relationship in a bottom-up manner and to help the rational design of ensemble materials with improved performance.