Abstract: To address the issue of high predator DNA contamination during the amplification of residual DNA in the stomach contents for the invasive hulong hybrid grouper with high stomach content rate,this study designed and tested three annealing blocking primers, and then applied to the analysis of the residual DNA in the stomach contents of the invasive hulong hybrid grouper. The results indicated that the blocking primer EFELblock2, which has the highest Melting Temperature(Tm) value, exhibited the best blocking effect at an annealing temperature of 56 ℃ and a concentration of 10 μM. The addition of blocking primers does not inhibit the amplification of DNA barcoding sequences from residual DNA of potential prey species in hulong hybrid grouper. High-throughput sequencing analysis showed that the addition of blocking primers significantly reduced the proportion of self-sequences in the invasive hulong hybrid grouper and did not affect the reconstruction of prey diversity information. The research results achieved the design and application of blocking primers for fish with empty stomachs in coral reef habitats, not only providing data support for the risk assessment of hulong hybrid grouper, but also laying an important foundation for further evaluating its negative impacts on marine ecosystems.