l-Tryptophan　decarboxylase　(TDC)　and　l-3,4-dihydroxyphenylalanine　decarboxylase　(DDC)　catalyze　the　decarboxylation　of　l-tryptophan,　5-hydroxytryptophan,　and　l-3,4-dihydroxyphenylalanine.　In　this　study,　we　analyzed　the　amino　acid　compositions　of　the　substrate-binding　pockets　of　TDC　from　Catharanthus　roseus　(CrTDC)　and　DDC　from　Sus　scrofa　(SsDDC),　explored　the　specificity　of　key　amino　acids　within　these　pockets,　and　elucidated　mechanisms　influencing　substrate　selectivity　and　catalytic　activity　in　both　enzymes,　using　whole-cell　catalysis　to　screen　mutants　and　determine　enzymatic　kinetic　parameters.　The　results　demonstrated　that　residues　Ala-103　and　Val-122　in　CrTDC,　along　with　their　corresponding　sites　Thr-82　and　Ile-101　in　SsDDC,　significantly　influence　substrate　selectivity　and　catalytic　efficiency.　Molecular　dynamics　simulations　revealed　that　substrate　selectivity　and　catalytic　efficiency　depends　on　the　nucleophilic　attack　distance　between　the　substrate＇s　amino　group　and　the　C4　＇　of　pyridoxal　5　＇-phosphate.　This　study　elucidates　the　catalytic　mechanisms　and　structural　bases　of　TDC　and　DDC,　guiding　enhancements　in　the　related　aromatic　monoamine　biosynthesis.