In order to accurately determine N-nitroso nornicotine (NNN) and its metabolites in rabbit blood, a high-performance liquid chromatography/tandem mass spectrometry method was established by optimizing chromatographic conditions and mass spectrometric conditions and using NNN-d4 as an internal standard. The samples were successively pretreated by protein precipitation, separated by a Waters-HILIC column, and analyzed by tandem mass detector under multi-reaction monitoring (MRM) mode. The results showed that the recoveries of NNN and its metabolites ranged from 80% to 111% with the relative standard deviations (RSDs) of 0.5% to 8.62% and the limits of detection of 0.039 to 0.217 ng/mL. This method is simple, sensitive, rapid, and suitable for the analysis of NNN and its metabolites in blood.