AbstractBACKGROUND: This paper describes a convenient and effective strategy to construct a highly sensitive amperometric biosensor for nitrite (NO2−) and hydrogen peroxide (H2O2). First, Pt nanoparticles (PtNPs) were electrodeposited on a glassy carbon electrode (GCE) surface, which promoted electron transfer and enhanced the loading of poly‐thionine (PTH). Subsequently, thionine (TH) was electropolymerized on the PtNPs/GCE, and gold nanoparticles (AuNPs) were assembled onto the PTH film to improve the absorption capacity of hemoglobin (Hb) and further facilitate electron transfer. Finally, Hb was immobilized onto the electrode through the AuNPs.RESULTS: Cyclic voltammetry (CV) and scanning electron microscopy (SEM) were used to characterize the fabrication process of the sensing surface. Under optimum conditions, the biosensors can be used for the determination of NO2− in the concentration range 70 nmol L−1 to 1.2 mmo L−1 and of H2O2 in the range 4.9 µmol L−1 to 6.8 mmol L−1. The detection limits (S/N = 3) were 20 nmol L−1 and 1.4 µmol L−1, respectively.CONCLUSION: The biosensor exhibits good analytical performance, acceptable stability and good selectivity. Copyright © 2011 Society of Chemical Industry