Hydroxytyrosol,　a　naturally　occurring　compound　with　antioxidant　and　antiviral　activity,　is　widely　applied　in　the　cosmetic,　food,　and　nutraceutical　industries.　The　development　of　a　biocatalytic　approach　for　producing　hydroxytyrosol　from　simple　and　readily　accessible　substrates　remains　a　challenge.　Here,　we　designed　and　implemented　an　effective　biocatalytic　cascade　to　obtain　hydroxytyrosol　from　3,4-dihydroxybenzaldehyde　and　l-threonine　via　a　four-step　enzymatic　cascade　composed　of　seven　enzymes.　To　prevent　cross-reactions　and　protein　expression　burden　caused　by　multiple　enzymes　expressed　in　a　single　cell,　the　designed　enzymatic　cascade　was　divided　into　two　modules　and　catalyzed　in　a　stepwise　manner.　The　first　module　(FM)　assisted　the　assembly　of　3,4-dihydroxybenzaldehyde　and　l-threonine　into　(2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic　acid,　and　the　second　module　(SM)　entailed　converting　(2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic　acid　into　hydroxytyrosol.　Each　module　was　cloned　into　Escherichia　coli　BL21　(DE3)　and　engineered　in　parallel　by　fine-tuning　enzyme　expression,　resulting　in　two　engineered　whole-cell　catalyst　modules,　BL21(FM01)　and　BL21(SM13),　capable　of　converting　30　mM　3,4-dihydroxybenzaldehyde　to　28.7　mM　hydroxytyrosol　with　a　high　space-time　yield　(0.88　g/L/h).　To　summarize,　the　current　study　proposes　a　simple　and　effective　approach　for　biosynthesizing　hydroxytyrosol　from　low-cost　substrates　and　thus　has　great　potential　for　industrial　applications.　©　2024　American　Chemical　Society.