In this paper, we report on the development of a mini-centrifuge-compatible microfluidic device that implements multistep liquid handling to realize bioassay protocols including enzymatic amplification steps such as enzyme-linked immunosorbent assay (ELISA). A device was developed by considering the geometry of the fluidic channel, the wettability of the microchannel, the volume and position of the chambers, and the entire assay process. The assay process includes injection of the sample into the reaction chamber, washing of the chamber, injection of the substrate, and injection of the stop buffer by two-mode (spin and stop) control that can be accomplished with an affordable minicentrifuge. During the assay protocol, both injection of the liquid into a chamber and draining of the liquid from the chamber are realized at a constant rotational speed, which is a key function for enzymatic amplification using a siphon-shaped channel. The injected liquid is held in the reaction chamber, both in the presence and absence of centrifugal force, then the liquid is drained once a liquid exceeding the critical volume is injected into the chamber. The reaction chamber was prepared for enzymatic amplification. The device was then applied to assay the avidin-biotin system, and the resulting assay performance was demonstrated to be comparable to or better than the conventional titer plate assay performance.