A tunable aptamer eleetrophoretic assay enables highly sensitive fluorescence detection of multiple proteins and protein isomers. The electrophoretic mobility of proteins is tuned with DNA aptamers binding to the target proteins. Fluorescently labeled aptamers of varying nucleotide lengths serve as both charge modulators for capillary electrophoresis separation and as fluorescent affinity probes for ultrasensitive laser-induced fluorescence detection. Simultaneous determination of pM levels of human immunodeficiency virus reverse transcriptase, thrombin, human immunoglobulin E, and two isomers of platelet-derived growth factor in dilute human serum demonstrates the potential applications of this assay to biomarker development.