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With the aggravation of sulfide pollution, more and more attention has been paid to the detection of sulfide in the environment. However, the detection of low-concentration sulfide is still a technical bottleneck to be solved urgently. In this study, a synergistic effect strategy that combines the co-catalysis of nanoporous gold (np-Au) and recombinant microbial cell with the excellent electrical conductivity of reduced graphene oxide (rGO) was proposed for the sensitive detection of low-concentration sulfide. A rGO/np-Au composite was fabricated and then used as an immobilization support for the bio-recognition element of recombinant Escherichia coli (E. coli) over-expressed sulfide: quinone oxidoreductase (SQR). A microbial biosensor (E. coli SQR/rGO/np-Au/GCE) was successfully constructed for the sensitive detection of low-concentration sulfide. Due to the synergistic effect of rGO, np-Au, and E. coli SQR cells, the sensitivity of the proposed microbial biosensor towards sulfide reached 400.42 μA mM−1 cm−2 with a wide linear response ranging from 100 nM to 7 mM, as well as a low detection limit of 98.5 nM using amperometric i-t curve method. Furthermore, the microbial biosensor was successfully applied to the detection of sulfide in wastewater with strong anti-interference ability, high reproducibility, and strong stability. These results confirmed that the proposed microbial biosensor was ideal for the detection of low-concentration sulfide in a reliable, specific, and sensitive way. • A sensitive microbial biosensor was successfully constructed for low-concentration sulfide detection. • The rGO/np-Au composite significantly improved the sensitivity of the microbial biosensor. • The reliable low-concentration sulfide detection in wastewater was successfully achieved. [ABSTRACT FROM AUTHOR]