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Toxic Chinese medicine residues in honey pose a serious threat to consumer health. <i>Gelsemium</i> is one of the nine ancient poisons, making the whole plant virulent. The residue of <i>Gelsemium</i> alkaloid in honey causes poisoning from time to time. Therefore, it is very important to establish a method for the detection of <i>Gelsemium</i> alkaloids in honey. In this study, a method of solid phase extraction (SPE) with two-dimensional liquid chromatography (2D-LC) was developed for the first time for the simultaneous determination of <i>Gelsemium</i> alkaloids in honey, including gelsemine, koumine and humantenmine. First, the honey samples were purified by a PRS cation exchange column and extracted with 5% ammoniated methanol. Then, we verified the methodological indicators, which were in line with the Codex Guideline requirements. The verification results are as follows: matrix-matched calibrations indicated that the correlation coefficients were higher than 0.998. The recovery was in the range of 81%–94.2% with an intraday precision (RSD) of ≤5.0% and interday RSD of ≤3.8%. The limit of detection for the three alkaloids was 2 ng/g. The limits of quantification for gelsemine and koumine were 5 ng/g, and humantenmine was 20 ng/g. This method can be applied to the monitoring of <i>Gelsemium</i> alkaloids in honey.