Guanine (G) and adenine (A) are two important components of nucleic acid and some small molecules in an organism. In this paper, we developed a new and convenient biosensor for the simultaneous determination of G and A based on overoxidized polypyrrole/multiwalled carbon nanotube and molybdenum disulfide modified glassy carbon electrode (PPyox/MWNTs-MoS2/GCE). The composite material exhibited remarkable electro-catalytic properties toward the oxidation ofGandAdue to its large surface area and fast electron transfer. The oxidation peak positions of G and A were at 0.73 V and 1.03 V, respectively, which allowed simultaneous detection of the two substances in coexistence solution. The designed biosensor exhibited linear responses to G and A both in the ranges 5-30 mu M and 30-120 mu M, with detection limits (S/N = 3) of 1.6 mu M (G) and 1.7 mu M (A), respectively. Moreover, interferences from some coexisting electro-active species including inorganic ions, glucose, glycine and citric acid were nearly negligible. Further, the sensing strategy was successfully used for the detection of G and A in real samples with satisfactory results, indicating a potential application for the simultaneous electrochemical detection of G and A. (c) 2019 The Electrochemical Society.