Human milk oligosaccharides (HMOs) are prebiotic compounds found in breast milk that favor the growth of beneficial bacteria in the infant gut. Often these compounds are derivatives of lactose or n-acetyllactosamine that contain either a fucose or sialic acid moiety. While there are many efforts underway to synthesize these beneficial complex carbohydrates, the current standard for their detection is low-throughput HPLC-MS. We present a high-throughput, fluorescence-based detection system for fucosylated and sialylated human milk oligosaccharides in vivo in E. coli. Such high-throughput systems facilitate directed evolution for increased in vivo HMO production. Additionally, the reporter enables the use of HMOs as orthogonal inducers of cellular circuits in synthetic biological applications.