As a green and powerful tool, biocatalysis has emerged as a perfect alternative to traditional chemistry. The bottleneck during process development is discovery of novel enzymes with desired properties and independent intellectual property. Herein, we have successfully bioprospected three novel bacterial alpha-l-rhamnosidases from human fecal metagenome using a combinatorial strategy by high-throughput de novo sequencing combined with in silico searching for catalytic key motifs. All three novel alpha-l-rhamnosidases shared low sequence identities with reported (< 35%) and putative ones (< 57%) from public database. All three novel alpha-l-rhamnosidases were over-expressed as soluble form in Escherichia coli with high-level production. Furthermore, all three novel alpha-l-rhamnosidases hydrolyzed the synthetic substrate p-nitrophenyl alpha-l-rhamnopyranoside and natural flavonoid glycosides rutin and naringin with some excellent properties, such as high activity in acidic pH, high activity at low or high temperature, and good tolerance for alcohols and DMSO. Our findings would provide a convenient route for target discovery of the promising biocatalysts from the metagenomes for biotransformation and biosynthesis.