The use of paper as a sensing platform has great potential for point-of-care diagnostics in low-resource settings. However, there is a need for approaches that enable a higher level of control and customisation of the paper properties and reduce the cost of production and immobilisation of bioreagents onto paper. This thesis explores whether introducing digital printing into papermaking in combination with the recombinant protein production of paper-binding bioreagents could provide an accessible, scalable tool for the fabrication of affordable paper-based microfluidic biosensors with customised and varying paper properties. A novel printing method based on the localised vacuum-driven filtration of fibre suspensions (‘vacuum-driven printing’) was explored for the direct formation and patterning of engineered paper. An analytical model based on incompressible cake filtration theory was derived to predict the final thickness of the printed paper as a function of printing parameters and showed good agreement with the experimental data. This model enabled the rational design of complex 2D paper patterns with controlled and varying thickness profile that could be formed in a single printing process. The recombinant fusion of a cellulose-binding domain (CBD) into the protein reagent enabled its one-step immobilisation and purification onto paper fibres directly from the crude lysate, significantly reducing the cost and number of downstream processing steps required for the production of the bioreagent. The specific immobilisation provided by the CBD helped retain the protein’s diagnostic activity during air drying and long-term dry storage, even after four months at 20-37°C, when compared to direct physisorption of the protein reagent onto paper. The protein-bound fibres could be directly patterned with the vacuum-driven printing technique into the detection zone of a paper-based assay, forming bioactive paper. A proof-of-concept paper-based microfluidic biosensor was fabricated in a single vacuum-driven printing process, using unmodified paper fibres to form microfluidic channels of varying thickness, and protein-bound fibres to form bioactive paper at the detection zone of the assay. This research demonstrated a simplified, accessible, and lower-cost pathway to the production of paper-based biosensors with customised properties.