Lateral flow assays (LFAs) provide a number of advantages as a detection platform, including simple capillary driven operation, portability, and low cost. However, they cannot handle multi-step reactions, including enzyme linked immunosorbent assay (ELISA), signal enhancement process, and a number of colorimetric reactions. To overcome this limitation of LFAs, a number of paper-based microfluidic devices that enable multi-step assays have been introduced. Nevertheless, complicated multi-step operation principle is required for multiple reagents loading. Although several paper-based microfluidic devices that perform one-step operation of multi-step assays have been proposed to improve complicated operation principle, their fabrication procedures still need to be improved. In this study, we developed a pressurized paper-based microfluidic device using both channel partition and delayed fluid flow by pressurizing nitrocellulose (NC) membrane. When the NC membrane is exposed to pressure, pores are collapsed and they play a role as a fluid resistance that results in delayed fluid flow. Furthermore, at a certain high pressure, pores are collapsed to an extent sufficient to prevent fluid flow so that collapsed pores can play a role as a channel partition. We characterized both delayed fluid flow and channel partition formation on pressurized NC membrane. By exploiting delayed fluid flow and channel partition formation, we developed a pressurized multi-channel immunostrip. Design of pressurized multi-channel immunostrip was optimized by controlling flow rate based on Darcy’s law. The pressurized multi-channel immunostrip consisted of three channels divided by two channel partitions and pressurized regions on the channels for delayed fluid flow. The immunostrip also had two detection regions for dual detection of target analytes. All the reagents re-quired for assay were pre-loaded and dried on the NC membrane. By one-step dipping the device into a sample solution, the reagents were sequentially rehydrated. Consequently, colorimetric signal for each analyte was developed by gold nanoparticle based immunocomplexes and colorimetric signal was amplified by gold enhancer that rehydrated by delayed fluid flow. As a proof of concept demonstration, we detected two kinds of fatal foodborne pathogens (Escherichia coli O157:H7 and Salmonella typhimurium) by signal enhancement. Detection limits of E. coli O157:H7 and S. typhimurium were about $10^5 CFU/mL$ and $10^6 CFU/mL$, respectively.