Fluorescence anisotropy (FA) holds great potential for multiplexed analysis and imaging of biomolecules since it can effectively discriminate fluorophores with overlapping emission spectra. Nevertheless, its susceptibility to environmental variation hampers its widespread applications in biology and biotechnology. In this study, we design FA DNA frameworks (FAFs) by scaffolding fluorophores in a fluorescent protein-like microenvironment. We find that the FA stability of the fluorophores is remarkably improved due to the sequestration effects of FAFs. The FA level of the fluorophores can be finely tuned when placed at different locations on an FAF, analogous to spectral shifts of protein-bound fluorophores. The high programmability of FAFs further enables the design of a spectrum of encoded FA barcodes for multiplexed sensing of nucleic acids and multiplexed labeling of live cells. This FAF system thus establishes a new paradigm for designing multiplexing FA probes for cellular imaging and other biological applications.