Bio-based production of D-1,2,4-butanetriol (BT) from renewable substrates is increasingly attracting attention. Here, the BT biosynthetic pathway was constructed and optimized in Escherichia coli to produce BT from pure D-xylose or corncob hydrolysates. First, E. coli BL21(DE3) was identified as a more proper host for BT production through host screening. Then, BT pathway was systematically optimized with gene homolog screening strategy, mainly targeting three key steps from xylonic acid to BT catalyzed by D-xylonate dehydratase (XD), 2-keto acid decarboxylase (KDC) and aldehyde reductase (ALR). After screening six ALRs, four KDCs and four XDs, AdhP from E. coli, KdcA from Lactococcus lactis and XylD from Caulobacter crescentus were identified more efficiently for BT production. The co-expression of these enzymes in recombinant strain BL21-14 led to BT production of 5.1 g/L under the optimized cultivation conditions. Finally, BT production from corncob hydrolysates was achieved with a titer of 3.4 g/L.