This paper presents a pressed paper-based dipstick that enables detection of foodborne pathogens with multistep reactions by exploiting the delayed fluid flow and channel partition formation on nitrocellulose (NC) membrane. Fluid behaviors are easily modified by controlling the amount of pressure and the position of pressed region on the NC membrane. Detection region of the dipstick is optimized by controlling flow rate and delayed time based on Darcy's law. All the reagents required for assay are dried on the NC membrane and they are sequentially rehydrated at the prepartitioned regions when the device is dipped into sample solution. In this manner, multistep reactions can be facilitated by one-step dipping of the dipstick into the sample solution. As a proof of concept, we performed detection of two fatal foodborne pathogens (e.g., Escherichia coli O157:H7 and Salmonella typhimurium) with signal enhancement. In addition, we expanded the utilization of channel partitions by developing a pressed paper-based dipstick into dual detection format.