We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 min of continuous signal enhancement, we can detect 4.4 pM human immunoglobulin E (IgE) and 9 pM human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT), which are among the lowest limits of detection (LOD) reported. IgE was detected in serum sample with a LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays.