Inosine monophosphate (IMP), as a critical umami substance, is one of the most important indicators for evaluating the quality of meat products. Here, a sensitive electrochemiluminescence (ECL) biosensor based on graphdiyne (GDY)/AuNPs/luminol nanocomposites was constructed to detect IMP. The GDY/AuNPs/luminol nanocomposites were synthesized by using simple one-pot method. GDY utilized its 2D framework to disperse and fix gold nanoparticles, which inhibited the agglomeration of gold nanoparticles and greatly improved its stability and catalytic properties. Importantly, GDY/AuNPs/luminol nanocomposites showed excellent catalytic ability and superior ECL activity towards luminol-H2O2 systems due to the synergistic effect of GDY and AuNPs. Under optimal conditions, the prepared biosensor exhibited a wide linear range from 0.01 g/L to 20 g/L, a satisfactory limit detection of 0.001 3 g/L, as well as an excellent specificity. Moreover, we carried out the precise analysis of IMP in actual meat samples with acceptable results compared to the liquid chromatography. We believe that this work could offer an efficient ECL platform for accurate and reliable report of IMP levels, which is significant for maintaining food quality and safety.