We have studied the construction of an electrochemical immunochromatography platform as one of the point-of-care testing devices from both experimental and theoretical aspects. In the electrochemical immunochromatography measurement, the layout of working, reference, and counter electrodes and the flow rate in a porous membrane are important in obtaining a highly sensitive and stable electrochemical signal. In this manuscript, we report the effects of the layout of the three electrodes and the elapsed time after sample injection on electrochemical signals. We found that the working electrode should be placed upstream of the reference electrode because an unfavorable electrochemical signal from a Ag+ ion from the reference electrode was observed on the working electrode when it was located downstream of the reference electrode. Furthermore, we found that the electrochemical signal was inversely proportional to the sixth root of time. Experimental and theoretical results are consistent with each other, exhibiting a high correlation coefficient of 0.99.