In this work, a novel electrochemical biosensor for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was developed based on cytochrome P450 2E1 (CYP2E1). The biosensor was fabricated by immobilization of CYP2E1 with poly (diallyldimethylammonium chloride) (PDDA) - poly (sodium-p-styrenesulfonate) (PSS) electrolyte film on a screen-printed carbon electrode (SPCE) electrodeposited with gold nanoparticles (AuNPs). The procedures of electrode modification were characterized by using scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). With the good conductivity of AuNPs as well as the immobilized CYP2E1, the electrochemical performances of the proposed biosensor for the detection of NNK showed a wide linear range of 0–386 μM with a sensitivity of 79 μA cm−2 mM−1 and a low detection limit of 7.71 μM. Additionally, excellent selectivity and reproducibility were also obtained. The fabricated NNK electrochemical biosensor was successfully employed in urine sample determination, exhibiting great potential in the real sample analysis.