The conventional neuraminidase inhibitor assay requiring complicated step-by-step operations was successfully integrated into a "single step" operation using a combinable poly(dimethylsiloxane) (PDMS) capillary (CPC) sensor. In the conventional neuraminidase inhibitor assay, complicated step-by-step operations including initial reaction of an inhibitor and an enzyme, and subsequent reaction with a fluorescent substrate are necessary. Furthermore, optimal pH conditions for the enzymatic reaction differ from those of fluorescence detection. Therefore, preparation of different pH buffer solutions is also essential. To simplify the neuraminidase inhibitor assay into a single-step operation, we applied our recently developed CPC sensor array. Because the CPC sensor allows independent immobilization of different reagents, such as enzymes and fluorescent substrates, simple introduction of an inhibitor solution by capillary action leads to fluorescence emissions. Here, we investigated the optimal pH that enables us to perform the neuraminidase inhibitor assay via a single step operation using the CPC sensor. When the reaction profile was investigated under the optimal pH, the total reaction time was shortened from 1 h to 15 min. Moreover, the neuraminidase inhibitor assay was validated using a typical inhibitor, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. The inhibition constant, Ki, which was calculated on the basis of the IC50 obtained from the inhibition curve, was in good agreement with the published values. Furthermore, we demonstrated a comparison of fluorescence responses for buffer solutions versus the solution of a well-known influenza drug, Relenza. We observed successful inhibition by Relenza. In addition to the CPC-based approach, the consumption of enzyme, substrate, and inhibitor was reduced to 1% of the amount used in conventional neuraminidase inhibitor assays.