cAMP(cyclic adenosine 3′,5′-monophosphate) and cGMP(cyclic guanosine 3′,5′-monophosphate) are ubiquitous second messengers which regulate myriads of functions in eukaryotic cells, and their signaling pathways are regarded as the popular target in functional food bioactives screening. In this review, we discuss the advantages and disadvantages of techniques such as radioisotope, homogeneous non-radioactive isotope and heterogeneous non-isotopic methods, which are used to measure cAMP and cGMP and describe how these techniques can be applied. The homogeneous non-radioactive isotope methods, including fluorescence resonance energy transfer technology (FRET), homogeneous time-resolved fluorescence resonance energy transfer technology (TR-FRET), polarization fluorescence detection technology (FP), are suitable for cAMP/cGMP high-throughput screening of bioactives for functional food based on its advantages of easy operation, high specificity and sensitivity.