In the last 10 years, paper-based electrochemical biosensors have gathered attention from the scientific community for their unique advantages and sustainability vision. The use of papers in the design the electrochemical biosensors confers to these analytical tools several interesting features such as the management of the solution flow without external equipment, the fabrication of reagent-free devices exploiting the porosity of the paper to store the reagents, and the unprecedented capability to detect the target analyte in gas phase without any sampling system. Furthermore, cost-effective fabrication using printing technologies, including wax and screen-printing, combined with the use of this eco-friendly substrate and the possibility of reducing waste management after measuring by the incineration of the sensor, designate these type of sensors as eco-designed analytical tools. Additionally, the foldability feature of the paper has been recently exploited to design and fabricate 3D multifarious biosensors, which are able to detect different target analytes by using enzymes, antibodies, DNA, molecularly imprinted polymers, and cells as biocomponents. Interestingly, the 3D structure has recently boosted the self-powered paper-based biosensors, opening new frontiers in origami devices. This review aims to give an overview of the current state origami paper-based biosensors, pointing out how the foldability of the paper allows for the development of sensitive, selective, and easy-to-use smart and sustainable analytical devices.