A sensitive, rapid and easy operated sensor for hydrogen peroxide (H2O2) determination in exhaled breath was developed in this study. The polyaniline (PANI) film and Cu nanoparticles (NPs) were successively electrochemical deposited on the surface of the three-dimensional nickel foam (NF), i.e., synthetized nano-Cu/polyaniline/NF. The Ni foam facilitates the charge and mass transfer, and it also provides a large amount of anchoring sites for Cu NP deposition. The synergistic electro-catalytic actions between Cu NPs and PANI highly enhanced the reduction current of H2O2. Under the optimized conditions, the linear range for monitoring H2O2 extends from 0.01 to 500 nM (R 2 = 0.998), and the limit of detection is 0.0026 nM. Integrating with collection of exhaled breath in a fritted midget bubbler, the developed sensor can clearly identify the difference of H2O2 levels between the smokers and non-smokers, as well as before and after smoking for these subjects. Its application performance indicates the developed sensor possesses a great potential for routine analysis of H2O2 in exhaled breath.