Lupeol is a pentacyclic triterpene that shows a variety of pharmacological properties. Compared to engineering the production of sesquiterpenes and diterpenes, it is much more challenging to engineer the biosynthesis of triterpenes in microbial platforms. This study showed our efforts on engineering the triterpene pathway in Escherichia coli and Saccharomyces cerevisiae cells by recruiting the codon-optimized three lupeol pathway genes from different organisms. By comparing their activities with their respective counterparts, the squalene synthase from Thermosynechococcus elongates (tSQS), the squalene epoxidase from Rattus norvegicus (rSE) and the lupeol synthase from Olea europaea (OeLUP) were introduced into E. coli BL21(DE3), a break-through from zero was observed for lupeol biosynthesis in a prokaryotic host. We also assessed the lupeol pathway under two different yeast backgrounds-WAT11 and EPY300, and have found that the engineered strains based on EPY300, named ECHHOe, processed the best lupeol-producing ability with the maximum lupeol titer being 200.1 mg l-1 at 30 °C in a 72 h-flask culture, which so far was the highest amount of lupeol obtained by a microbial system and provides a basis for further industrial application of lupeol in the future.