Conventional dietary assessments are challenging in hematophagous species, particularly in sea lamprey (Petromyzon marinus). However, recent technological developments and molecular approaches have provided an attractive alternative through the use of DNA metabarcoding. While DNA metabarcoding has been used for dietary analyses in numerous species, including lampreys, applications of universal primers that detect a diverse set of prey items can be limited by the amplification of predator DNA. In this study, we designed and tested eight blocking primers designed to suppress the amplification of sea lamprey DNA with vertebrate-universal primers targeting the mitochondrial 12S rRNA gene. This approach allowed for the use of a single marker to amplify a taxonomically diverse suite of host species, in contrast to previous studies that used multiple taxon-specific primer pairs (e.g., Salmonidae, Cyprinidae, and Catostomidae). Candidate blocking primers evaluated in this study differed in base pair length, end sequence modification, and purification method. Samples with different sea lamprey-to-host DNA ratios were subjected to multiple detection methods including gel electrophoresis, quantitative PCR, and DNA metabarcoding to assess the ability of each blocking primer to selectively suppress amplification of the sea lamprey 12S gene region. All blocking primers tested performed well and demonstrated high effectiveness, suppressing sea lamprey reads by > 99.9% in mock communities and improving host DNA sequence recovery across various sample types, including wild-caught lamprey. Results show that the blocking primers evaluated can facilitate molecular diet analysis in sea lamprey, allowing the amplification of a taxonomically diverse range of host fish species with universal primers.