In the present study, for the first time, fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. FRET signal measures the interaction between two molecules labeled with two different fluorophores (i.e., the donor and the acceptor), by the transfer of energy from the excited donor to the acceptor. Various methods are available in the extent literature to quantify and plot the FRET signal27, but the measurement that they offer involves numerous practical difficulties, including calculation error, difficult interpretation, and high sensitivity. We propose here a quantitative method by using a non-fabricated parchment paper surface to measure the FRET system with controlled amounts (nanoliter volume) of donor and acceptor fluorophores using a conventional inkjet printer equipped with four cartridges. The reaction sample solutions, including cAMP, PDE4B, roliparm or roflumilast, Eu- anti cAMP, and ULight cAMP are sequentially printed on parchment paper through a layer-by-layer process. This paper demonstrates successful completion between Eu chelate- labeled cAMP tracer (donor) and ULight- anti-cAMP dye (acceptor) on parchment paper. After printing, Eu chelate- labeled cAMP tracer is excited, and the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm using a fluorescent microscope. In the absence of free cAMP maximum, the FRET signal was achieved on paper, while a decrease in the FRET signal was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. Parchment papers are found to be a unique substrate to measure fluorescent energy transfer, due to their insignificant self-absorption, which facilitates efficient interaction of reaction components. This new parchment paper-based enzyme-inhibitor interaction/FRET assay offers several major advantages, including low reagent consumption, cost-effectiveness, no need of a fabrication process, stability, and biodegradability. In contrast to standard FRET measurements, the inkjet printing-based FRET determination precisely evaluates the inhibitory mole 50 (IM50) with an effective number of mole of PDE4B inhibitor printed per spot. IM50 is the amount of inhibitor used to react with different concentrations of substrate printed on paper. Hence, the methodology reported here constitutes an innovative approach towards the quantitative determination of FRET signals generated on paper.