An electrochemical microfluidic device with two sensing sites in the upper and lower streams of a flow channel was fabricated to measure the K-value as a means of evaluating the freshness of fish. In this device, plugs of solutions were processed using mechanisms to place a plug at the sensing site and to merge and mix two plugs in a single flow channel. The sums of ATP-related compound concentrations used for the calculation of the K-value were measured at the first and second sensing sites. The ratio of the output currents agreed well with the K-value calculated from predetermined concentrations in standard solutions. The K-values of jack mackerel, yellow tail, and sea bream extracts were then obtained using the device and were found to agree well with those obtained by high-performance liquid chromatography (HPLC). In addition, the changes in the K-value with time were observed to depend strongly on the type of fish for these three fish extracts.