In this work, we developed a novel strategy using fluorescent quantum dots (QDs) combined with molecularly imprinted polymers (MIPs) on three-dimensional (3D) origami paper-based microfluidic devices for specific recognition and sensitive detection of phycocyanin. This method can realize the liquid phase of QDs@MIPs being transferred to the solid-phase paper base and achieve easy portability for the analysis. Under optimal conditions, we successfully demonstrated the proposed paper@QDs@MIPs 3D microfluidic chip for the sensitive and selective detection of phycocyanin protein target in a simple and robust manner. Our results revealed that the method exhibited a dynamic response to phycocyanin in the range of 10–50 mg/L with a limit of detection of 2 mg/L. Importantly, this device could provide quantitative information very conveniently and show great potential to be further extended to the detection of other proteins or biomarkers for environmental and food safety research.