Conjugation of biomolecules to colloidal nanoparticles, such as quantum dots (QDs), often leads to change in mobility. We discover that linking DNA molecules to quantum dots alters their surface charge density without significantly increasing the hydrodynamic radius, causing a prominent shift in electrophoretic mobility. In this study, a high-resolution molecular quantification method named quantification by QDs electrophoretic mobility shift (qQEMS) is developed based on the charge-dominant transformation that closely associates DNA quantity to QDs electrophoretic mobility. The versatility of qQEMS is demonstrated by a number of quantification assays in which DNA molecules functioned as enzyme substrates, target-specific probes, and competitive charge carriers. qQEMS shows a great potential as a generic and versatile quantification platform for a wide range of applications.