Testosterone　(TS),　an　important　hormone　pharmaceutical　and　precursor　for　the　synthesis　of　other　steroids,　is　traditionally　produced　by　a　polluting　and　costly　multistep　chemical　synthesis.　Producing　TS　by　a　biological　method　is　eco-friendly,　while　its　application　is　still　limited　by　its　low　substrate-loading,　low　conversion,　long　duration,　and　low　time-space　yield.　Focusing　on　these　problems,　a　new　enzyme　system　was　constructed　in　which　the　carbonyl　reductase　PmCR　was　employed　to　reduce　4-androstene-3,17-dione　(4-AD)　into　TS　for　the　first　time　coupled　with　the　formate　dehydrogenase　BstFDH_m.　Based　on　molecular　dynamics　(MD)　simulation,　the　enzyme　system　was　upgraded　by　rationally　designing　PmCR,　and　the　variant　L136S　was　screened　with　a　14-fold　enhanced　catalytic　efficiency　kcat/Km　and　the　highest　performance　in　4-AD　reduction.　The　testosterone　production　of　the　L136S-BstFDH_m　system　was　further　enhanced　by　condition　optimization　and　competence　to　synthesize　TS　with　the　highest　reported　4-AD　loading　(28.8　g/L),　conversion　(100%),　and　testosterone　time-space　yield　(2.90　g/L/h)　using　a　microbial　method,　highlighting　its　promising　application　in　TS　synthesis.　The　successful　attempt　in　this　study　not　only　provides　a　promising　biocatalyst　and　efficient　approach　for　TS　green　synthesis　but　also　opens　the　door　to　the　application　of　carbonyl　reductase　in　the　green　synthesis　of　diverse　pharmaceutical　sterols.　©　2022　American　Chemical　Society.