Plants　have　an　extensively　large　number　of　enzymes　including　glycosyltransferases　that　are　important　in　the　biosynthesis　of　natural　products.　However,　it　is　time-consuming　and　challenging　to　study　these　enzymes　and　only　a　small　percentage　of　them　have　been　well-characterized.　Here,　we　report　a　rapid　method　to　screen　plant　glycosyltransferases　using　a　linear　DNA　expression　template　(LET)　based　cell-free　transcription-translation　system　(TX-TL).　As　a　proof　of　concept,　we　amplified　and　tested　glycosyltransferases　from　Arabidopsis　thaliana　and　showed　that　the　catalytic　activity　results　of　these　glycosyltransferases　from　LET-based-TX-TL　were　consistent　with　previous　studies.　We　then　chose　a　local　medicinal　plant　Anoectochilus　roxburghii,　acquired　its　transcriptome　sequences,　and　applied　this　method　to　study　its　glycosyltransferases.　We　rapidly　expressed　all　the　putative　UDPglucose　glycosyltransferases　using　LET-based-TX-TL　and　discovered　6　unreported　active　glycosyltransferases　which　can　catalyze　the　glycosylation　of　quercetin　into　isoquercitrin.　Thus,　LET-based-TX-TL　was　shown　to　be　a　powerful　tool　for　researchers　to　rapidly　screen　plant　glycosyltransferases　for　the　first　time.