Abstract A novel bioelectronic sniffer for nicotine in the gas phase was developed with enzyme inhibition principle to butyrylcholinesterase activity. The bioelectronic devices for nicotine in the gas and liquid phases were constructed using a Clark-type dissolved oxygen electrode and a membrane immobilized butyrylcholinesterase and choline oxidase. After the assessment of the sensor performances to choline and butyrylcholine as pre-examinations, the characteristics of the biosensor and bio-sniffer for nicotine were evaluated in the liquid and gas phases, respectively. The sensor signal of the bio-devices with 300 μmol l −1 of butyrylcholine decreased quickly following application of nicotine and reached to the steady-state current, thus relating the concentration of nicotine in the liquid and gas phases. The biosensor was used to measure nicotine solution from 10 to 300 μmol l −1. In the gas-phase experiment, the current signal of the bio-sniffer was also found to be linearly to the nicotine concentration over the range of 10.0–1000 ppb including 75.0 ppb as threshold limit value (TLV) by American Conference of Governmental Industrial Hygienists (ACGIH).