In this paper, a 3D microfluidic origami device combined with electrochemiluminescence (ECL) immunosensor was introduced for sensitive point-of-care testing of carcinoma antigen 125 in clinical serum samples for the first time. This microfluidic origami device was fabricated by directly screen-printing carbon working, counter electrodes and Ag/AgCl reference electrode including their conductive pads on wax-patterned pure cellulose paper, and was activated by folding to form a 3D electrochemical cell. To construct a sensitivity-enhanced sandwich-type ECL immunosensor, gold nanoparticles (AuNPs) was synthesized to modify the working electrode to provide a good pathway of electron transfer and enhance the immobilized amount of capture antibody (McAb 1 ), and luminol functionalized gold nanoparticles (Lu-AuNPs) was synthesized to label signal antibody (McAb 2 ). With the aid of a facile device-holder, cyclic voltammetry was applied on microfluidic origami ECL immunodevice to trigger ECL. Under optimal conditions, this microfluidic origami ECL immunodevice provided a good linear response range from 0.01 to 100UmL −1 with a detection limit of 0.0074UmL −1 . The proposed ECL immunodevice presented high sensitivity and stability, and could be applied in point-of-care testing of other tumor markers for remote regions and developing countries.