A new method is developed whereby the enantiomeric composition of a chiral analyte is determined by steady-state fluorescence anisotropy. A theoretical model is presented showing that the measured anisotropy of an enantiomeric mixture in the presence of a chiral selector is dependent on the selectivity, the concentration of free selector, and the enantiomeric composition. Furthermore, for a given system the relationship between the measured anisotropy and the enantiomeric composition is predicted to be linear. The prediction of a linear relationship was confirmed experimentally by examining mixed enantiomeric compositions of 1,1'-binaphthyl-2,2'-diylhydrogen phosphate in the presence of beta- and alpha-cyclodextrin. The enantiomeric compositions of four solutions of mixed enantiomers were determined based on a 2-point calibration with an average absolute error of less than 2%.