Novel 3D barcodes with an extraordinarily high encoding capacity are developed through a flexible, accurate, and reproducible method. Here, the three dimensions refer to the size, fluorescence emission wavelength, and intensity of the barcodes. As a proof of concept, a 3D barcode library with an encoding capacity of 100 flow cytometry-distinguishable barcodes is achieved successfully by combining the multiscaled host particles with a set of guest nanoparticles owning to their individual different fluorescent intensity via an ingenious host-guest structure, where fluorescein isothiocyanate and a kind of quantum dots are separately loaded inside. Five-plexed tumor marker detection is further implemented, and the results demonstrate the strong feasibility of 3D barcodes in multiplex assays. [ABSTRACT FROM AUTHOR]