A highly sensitive enzyme inhibition analytical platform was established firstly based on paper-supported 3D interwoven AuPd-reduced graphene oxide (rGO) network (NW) for real-time and in situ analysis of HS released from cancer cells. The novel paper working electrode (PWE) with large electric conductivity, effective surface area and unusual biocompatibility, was fabricated via controllably assembling rGO and AuPd alloy nanoparticles onto the surface of cellulose fibers and into the macropores of paper, which was employed as affinity matrix for horseradish peroxidase (HRP) loading and cells capture. It was the superior performances of AuPd-rGO-NW-PWE that made the loaded HRP exhibit excellent electrocatalytic behavior to HO, bring the rapid enhancement of current response. After releasing HS, the current response would be obviously decreased due to the efficient inhibition effect of HS on HRP activity. The inhibition degree of HRP was directly proportional to the amount of HS, and so, the flux of HS released from cells could be recorded availably. Thus, this proposed enzyme inhibition cyto-sensor could be applied for efficient recording of the release of HS, which had potential utility to cellular biology and pathophysiology.