In order to explore the molecular mechanism of alkaloid biosynthesis in lotus leaves, metabolomics and transcriptomics sequencing analyses were performed on mature lotus leaves of ‘Taikong Lian’ (high alkaloid content), ‘Juwuba’ (medium alkaloid content) and ‘Dazu Honglian’ (low alkaloid content) cultivars with significant differences in alkaloid content. Metabolomics analysis showed that there were 30, 32 and 14 different metabolites in the three groups of ‘Dazu Honglian’ vs ‘Taikong Lian’ (low vs high alkaloid content), ‘Dazu Honglian’ vs ‘Juwuba’ (low vs medium alkaloid content), and ‘Taikong Lian’ vs ‘Juwuba’ (high vs medium alkaloid content), respectively. These differential metabolites were mainly three types of isoquinoline alkaloids, namely benzylisoquinoline, bis-benzylisoquinoline and aporphine alkaloids, specifically including caaverine, 3\mathrm{\prime }-glucosyl-6, 7-dihydroxy-N-methyl-benzyltetrahydroisoquinoline, dopamine, L-tyramine, etc. To further explore the key genes of the above isoquinoline alkaloid biosynthesis pathway, the transcriptomics sequencing analysis of three cultivars were performed. The numbers of differentially expressed genes (DEGs) among the three groups (‘Dazu Honglian’ vs ‘Taikong Lian’, ‘Dazu Honglian’ vs ‘Juwuba’, and ‘Taikong Lian’ vs ‘Juwuba’) were 2 866, 2 739 and 3 932, respectively; and there were 379 DEGs in common, which contained isoquinoline alkaloid biosynthesis pathway genes. Combined with the results of metabolomics analysis, six key DEGs, including NnCYP80G, Nn6OMT, NnTYDC, NnNCS, NnRAV and NnERF, were finally screened and verified by real-time fluorescent quantitative polymerase chain reaction, which can be used for subsequent gene function verification and molecular regulation network analysis.