A rapid, environmental friendly, and sensitive biosensor for detection of zearalenone (ZEN) was developed at glassy carbon electrode (GCE) using nanoporous bimetallic alloy as label of secondary antibody (Ab(2)). The biosensor was prepared by immobilizing nitrogen-doped graphene sheets (N-GS) captured primary antibody (Ab(1)) on GCE. The analytes were then bound to Ab(1) for further capture of antibodies, labeled by the nanoporous bimetallic alloy, nanoporous PtCo alloy (NP-PtCo). NP-PtCo with three-dimensional bicontinuous spongy structures and various hollow channels was prepared by corrosion of PtCoAl alloy, and exhibited remarkably improved electrocatalytic activity toward H2O2. Within ZEN concentration range (0.005-25 ng mL(-1)), the biosensor exhibited a highly sensitive response to ZEN with a detection limit of 2.1 pg mL(-1). Several factors that would affect the performance of the biosensor were studied, such as erosion time of NP-PtCo, concentration of NP-PtCo, pH of buffer solution, and concentration of N-GS. After H2O2 was injected into the buffer solution, the amperometric response of the biosensor can be used to detect ZEN by chronoamperometry. The developed biosensor shows good selectivity and sensitivity to ZEN and is promising in feed safety analysis. (C) 2013 Elsevier Ltd. All rights reserved.