In analytical chemistry, preconcentration represents a critical step able to enhance the accuracy of detection; however, the experimental procedures needed to preconcentrate samples might be characterized by drawbacks regarding the whole analytical process, e.g., being complex, invasive, and/or time-consuming. In this study, a novel 3D paper-based origami device is introduced for multifold analyte preconcentration. Leveraging the benefits of paper-based substrates, the proposed architecture boosts sample preconcentration while minimizing time and tasks for measurements, solely by exploiting the porous and versatile nature of paper-based substrates. In comparison with other paper-based approaches reported in the literature for preconcentration, the present architecture offers the ability to be programmed for obtaining the needed sensitivity increase without sacrificing measurement time. To demonstrate the efficacy of the novel approach, the 3D paper-based origami device was deeply characterized, including the most relevant parameters, i.e., disk size and number, unfolding time, and volume, and subsequently applied for the preconcentration and the detection of various analytes in real matrices, namely, mercury in tap water and glucose in sweat, resulting in a 400% and 300% sensitivity enhancement, respectively. This innovative preconcentration tool addresses the limitations of existing conventional methods, providing increased sensitivity without the use of expensive and time-consuming procedures through only exploiting the intrinsic properties of paper-based substrates and a rationale design. The proposed architecture emerges as a universal tool to be adopted and programmed for various analytical systems and fields of application.