BackgroundPerilla frutescensis widely used as both a medicine and a food worldwide. Its volatile oils are its active ingredients, and, based on the different volatile constituents,P. frutescenscan be divided into several chemotypes, with perilla ketone (PK) being the most common. However, the key genes involved in PK biosynthesis have not yet been identified.ResultsIn this study, metabolite constituents and transcriptomic data were compared in leaves of different levels. The variation in PK levels was the opposite of that of isoegoma ketone and egoma ketone in leaves at different levels. Based on transcriptome data, eight candidate genes were identified and successfully expressed in a prokaryotic system. Sequence analysis revealed them to be double bond reductases (PfDBRs), which are members of the NADPH-dependent, medium-chain dehydrogenase/reductase (MDR) superfamily. They catalyze the conversion of isoegoma ketone and egoma ketone into PK in in vitro enzymatic assays. PfDBRs also showed activity on pulegone, 3-nonen-2-one, and 4-hydroxybenzalacetone. In addition, several genes and transcription factors were predicted to be associated with monoterpenoid biosynthesis, and their expression profiles were positively correlated with variations in PK abundance, suggesting their potential functions in PK biosynthesis.ConclusionsThe eight candidate genes encoding a novel double bond reductase related to perilla ketone biosynthesis were identified inP. frutescens, which carries similar sequences and molecular features as theMpPRandNtPRfromNepeta tenuifoliaandMentha piperita, respectively. These findings not only reveal the pivotal roles of PfDBR in exploring and interpreting PK biological pathway but also contribute to facilitating future studies on this DBR protein family.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04345-1.