Rod-like Sb2Se3 material prepared by a simple hydrothermalmethodwas dispersed with ionic liquid (IL) to construct Sb2Se3/IL/GOx-CPE and Sb2Se3/IL/Cyt c-CPE for the detection of glucose and nitrate (NO2-), respectively. Especially, the effect of preparation condition like reaction time on electrochemistry performance was discussed. The proposed biosensor showed excellent performances with wide linear range of 8-1700 mu M and the detection limit of 2.0 mu M for glucose, and 5-5300 mu M and 1.2 mu M for NO2-, respectively. Moreover, in the chain-type crystal structure of Sb2Se3, strong covalent bond along the c crystallographic axis and much weaker van der Waals bonding between the chains of the layered structure were existed, which facilitated the formation of rod-like Sb2Se3. This was why the rod-like Sb2Se3 could be used to immobilize GOx and Cyt c. With those remarkable advantages such as low cost, satisfactory reproducibility, stability and reliability, the V-VI binary chalcogenides such as rod-like Sb2Se3 material could be an option sensing platform for the extensive application of biosensors. (C) 2016 The Electrochemical Society. All rights reserved.