“One stone–three birds”: we developed a smart portable electrochemical sensor based on electrodeposited ferrocene-functionalized multiwalled carbon nanotubes (FC-MWCNTs) for the in vitro and in vivo detection of nicotine (NIC) in tobacco samples. The “one stone” was the establishment of the standard curve for the concentrationof NIC based on the oxidation of NIC itself as a single signal on the FC-MWCNT modified electrode or to use the FC-MWCNTs as a ratiometric probe to establish the standard curve for the NIC concentration on the modified electrode. The “three birds” were: (1) to broaden the application of the Nernst equation (the correlation between the potential and the concentration); (2) to use the function of materials modified by electrodeposition from multiple perspectives; and (3) to use a portable electrochemical workstation with a smartphone as an intelligent electrochemical sensing platform for NIC detection. The Tyndall effect, scanning electron microscopy, X-ray diffraction, UV-vis spectrophotometry, Fourier transform infrared spectrophotometry, Raman spectroscopy, cyclic voltammetry and electrochemical impedance spectrometry were used to characterize the properties of the sensor materials. The effects of the electrochemical properties, such as the electrochemical active area, the charge transfer resistance and the adsorbed charge were investigated. The sensor was then studied by differential pulse voltammetry. Under the optimized conditions, the detection range of NIC was 60–1000 μmol L⁻¹ and the detection limit was 4.25 μmol L⁻¹ in the single-signal mode and 0.44 μmol L⁻¹ in the ratiometric signal mode. The sensor can be used for the rapid and accurate detection of NIC in tobacco samples, both in vitro and in vivo.