Herein, we designed four peptides appended with different numbers of histidine (His(n)-peptide). We launched a systematic investigation on quantum dots (QDs) and His(n)-peptide self-assembly in solution using fluorescence coupled CE (CE-FL). The results indicated that CE-FL was a powerful method to probe how ligands interaction on the surface of nanoparticles. The self-assembly of QDs and peptide was determined by the numbers of histidine. We also observed that longer polyhistidine tags (n6) could improve the self-assembly efficiency. Furthermore, the formation and separation of QD-peptide assembly were also studied by CE-FL inside a capillary. The total time for the mixing, self-assembly, separation, and detection was less than 10 min. Our method greatly expands the application of CE-FL in QDs-based biolabeling and bioanalysis.