The affinity of boronic acids to cis-diol-containing biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Although a few analytical tools have been available for the characterization of the interactions, these techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diol-containing biomolecules. Therefore, a widely applicable method is still greatly needed. In this work, an affinity capillary electrophoresis (ACE) method was established and validated to probe the interactions between boronic acids and cis-diol-containing biomolecules. The method was proven to be applicable to almost all types of cis-diol-containing biomolecules and boronic acids. Based on this method, a quantitative, comparative study on the interactions between 14 boronic acids that have important potentials for application with 5 typical monosaccharides of biological importance was carried out. The findings provided new insights into boronate affinity interactions, particularly the relationship between the binding strength with the molecular structures of the binding species. Besides, effects of pH and temperature on the binding strength were also investigated. This method exhibited several significant advantages, including (1) possibility of simultaneous study of multiple interactions, (2) low requirement on the purity of the binding species, (3) wide applicability, and (4) high accuracy and precision.