Molecular imprinting technique is introduced into microfluidic paper-based analytical devices (μ-PADs) through electropolymerization of molecular imprinted polymer (MIP) in a novel Au nanoparticle (AuNP) modified paper working electrode (Au-PWE). This is fabricated through the growth of a AuNP layer on the surfaces of cellulose fibers in the PWE. Due to the porous morphology of paper as well as the high specific surface area and conductivity of the resulting AuNP layer on the cellulose fibers, the effective surface area and the sensitivity of the Au-PWE is enhanced remarkably. Based on this novel MIP-Au-PWE and the principle of origami, a microfluidic MIP-based electro-analytical origami device (μ-MEOD), comprised of one auxiliary pad surrounded by four sample tabs, is developed for the detection of D-glutamic acid in a linear range from 1.2 nM to 125.0 nM with a low detection limit of 0.2 nM. The selectivity, reproducibility, and stability of this μ-MEOD are investigated. This μ-MEOD would provide a new platform for high-throughput, sensitive, specific, and multiplex assay as well as point-of-care diagnosis in public health, environmental monitoring, and the developing world.