RNA isolation from the hepatopancreas of adult abalone is challenging, as indicated by the variable results of reverse transcription-polymerase chain reaction (RT-PCR) amplification. In this study, three RNA isolation methods were tested to evaluate their usefulness in RNA isolation from various abalone tissues. Unlike the conventional method (method I), methods II and III included additional centrifugation and lithium chloride precipitation steps, respectively. RNA quality assessment based on the RT-PCR amplification of ribosomal protein L3 (RPL3) showed little difference in the quality of RNA isolated from muscle, regardless of the isolation method used. While RNA isolated from the hepatopancreas using methods I and II resulted in lower levels of RPL3 amplification, method III was found to be a more effective method of RNA extraction from the hepatopancreas, as indicated by a 30–60-fold increase in the RPL3 level. Competitive inhibition experiments using mixtures of RNA prepared by each method were performed to investigate which RT-PCR step was inhibited by materials present in the RNA preparation. An almost complete absence of RPL3 amplification was observed in RT-PCR with a cDNA template prepared from a mixture of RNAs containing a small amount of hepatopancreatic RNA isolated using method I. A drastic decrease in the incorporation of a fluorescence-labeled nucleotide into cDNA was also evident in the reverse transcription containing hepatopancreatic RNA isolated using method I. These results indicate a distinct inhibition of cDNA synthesis by materials present in the hepatopancreatic RNA preparation isolated using method I, but not present when method III is used. A competitive inhibition experiment was performed using mixtures of cDNA prepared from RNA isolated by each method to investigate whether the materials used in the preparation inhibited the polymerase chain reaction (PCR) step. The level of RPL3 amplification was proportional to the amount of cDNA prepared from hepatopancreatic RNA isolated using method III. The comparable degree of amplification regardless of the amount of cDNA prepared from RNA isolated using method I, together with the low cDNA synthesis in the latter, indicated that it had little, if any, inhibitory effect on the PCR step. This study reports an RNA isolation method that is applicable to the hepatopancreas and other abalone tissues with similar efficacy.