In order to solve the high predator DNA contamination problem during the amplification of residual DNA in the stomach contents of invasive hulong hybrid grouper with high gastric emptying rate, in the report, three annealing blocking primers were designed, tested, then, the primers were used for 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 (T_m) value, exhibited the best blocking effect at an annealing temperature of 56 ℃ and a concentration of 10 μmol·L⁻¹. The addition of blocking primers did not inhibit the amplification of DNA barcoding sequences from residual DNA of potential prey species in hulong hybrid grouper. High-throughput sequencing analysis results 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. Our findings provided the data support for the risk assessment of hulong hybrid grouper, and laid an important foundation for further evaluating its negative impacts on marine ecosystems.