© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimDeveloping a facile approach for the manipulation of the direction and order of the enzymatic reactions via sequential immobilization on inexpensive substrates is a continuous demand. Herein, a new methodology is introduced that allows making a desired enzymatic reaction pathway on a paper-based microfluidic-membrane based biosensor (P-µMB). Although the method is universal, here, as a proof-of-concept, the sequential immobilization of α-amylase, glucose oxidase (GOx) and horseradish peroxidase (HRP) is presented for fabricating a P-µMB. To this end, hydrophilic polydopamine/polyethyleneimine patterns are created on the hydrophobic polypropylene membrane using 3D printing and a polydimethylsiloxane (PDMS) mold, and a coating layer of silver nanoparticles (AgNPs) is used to modify the patterns. The enzymes are then individually immobilized on the desired locations with another set of PDMS molds. It is observed that AgNPs P-µMB in the sequential immobilization system has stable activity at various temperature and pH regimes, high selectivity toward starch, wide-range linear sensitivity, and a limit of detection of 0.002% w/w starch. A smartphone camera is used for the quantitative analysis of the analyte with the mean gray intensity as the analytical parameter. This developed system provides a platform for further sequential immobilization of other types of biological elements.