A stationary liquid-phase lab-on-a-chip (SLP-LOC), in which magnetic particles are used as a solid phase, has been previously reported as a novel platform for immunoassay methods. The SLP-LOC had a simple structure with two chambers connected by a narrow channel. The assay was performed by transporting the particles from one chamber to the next while the liquid phases were in a stationary state. However, each chamber was frequently contaminated by other chamber materials because of the small volume of the channel. Consequently, in this work, we have developed a three-chamber SLP-LOC (3C-LOC) featuring an additional chamber containing a buffered washing solution positioned between the existing two chambers and connected by channels. The dimensions of the 3C-LOC were optimized to fit into a standard spectrophotometer cuvette holder, allowing the direct measurement of absorbance. The 3C-LOC exhibited improved washing efficiency, reliability, and sensitivity in an assay of standard saxitoxin samples. Furthermore, the method was used to effectively determine whether paralytic shellfish toxin (PST) levels in shellfish samples exceeded regulatory limits. The results of this study indicate that the 3C-LOC could provide a simple and rapid method to monitor PSTs in the field.