In our previous work, we observed a gradient increase in the volume used to print per spot according to the gradient increase of the printing setting32. Here, the C cartridge was filled with PDE4B inhibitors and the C value was set as the gradient range, and thus the gradient number of moles of roliparm/roflumilast was printed on parchment paper. In contrast, a decrease in fluorescent signal was recorded as the number of mole of inhibitor increased per spot. Paper-based inkjet printing-PDE4B inhibitory assay verifies that 0.29 × 10−13 to 2.72 × 10−13 mole of roliparm/ 0.04 × 10−13 to 2.5 × 10−13 mole of roflumilast (PDE4B inhibitor) printed per spot effectively interacts with 2.81 × 10−13 mole of PDE4B enzyme and 2.79 × 10−16 mole of cAMP, which permits a linear reduction in fluorescent signal. Consequently, from the observed results, it is evident that paper-based inkjet printing-PDE4B inhibitory analysis ensures specific and rapid FRET determination on paper. Compared to conventional well plate method, inkjet printing-based FRET signal determination utilizes a lesser reaction volume up to the nL range. The number of mole of PDE4B inhibitor reacting with PDE4B enzyme significantly verified this, which was four-orders less than the standard assay method. In the presence of cAMP, PDE4B, roliparm/ roflumilast, Eu-cAMP tracer and ULight-anti-cAMP; PDE4B enzyme activity were inhibited by roliparm/roflumilast, while Eu-cAMP recognizes free cAMP. Hence, Eu-cAMP is less available to bind with ULight-anti-cAMP. Conversely, the level of energy transferred to ULight was decreased. Therefore, a significant reduction in fluorescent signal was recorded as a function of the increase in the number of mole of PDE4B inhibitor reacted on paper. The methodology reported here facilitates an innovative approach towards the determination of FRET signals generated on paper. Since this system is relatively inexpensive, readily available and involves simple operating procedures, non-professionals can effectively perform the analysis.