Objective: To separate a new important component 5-Hydroxymethyl-2-furfural (5-HMF) in cigarette smoke and provide material guarantee for evaluating its role in the respiratory system injury caused by smoking. Methods: A combination of liquid-liquid extraction and sephadex LH-20 column chromatography was used to extract and purify 5-HMF from the cigarette smoke extract (CSE) dissolved in chloroform. First, the total extract of cigarette smoke (CSE) was subjected to two consecutive liquid-liquid extractions with double-distilled water to transfer 5-HMF from the chloroform phase to the aqueous phase. Then, sephadex LH-20 was employed to separate 5-HMF from the aqueous phase. The efficiency of the extraction and separation processes was determined using high performance liquid chromatography-ultraviolet detection (HPLC-UV). GraphPad Prism software was used for two-way analysis of variance (Two-way ANOVA) and Tukey’s multiple comparisons test to evaluate the stability and reproducibility of the method. Results: The two-step liquid-liquid extraction transferred approximately 92.18% of 5-HMF from CSE to the aqueous phase. sephadex LH-20 enabled the separation and recovery of approximately 94.88% of 5-HMF from the aqueous phase.The coefficient of variation (CV) was 8.53% across three independent experiments. Two-way ANOVA indicated that although minor differences existed among different cigarette samples (P < 0.01), the elution profile of 5-HMF was highly consistent (tube number factor: P < 0.0001), accounting for 97.04% of the total variation, confirming the good reproducibility of the method. Based on the content calculation, this method achieved the separation of approximately 89.55% of 5-HMF from CSE, while the traditional component nicotine in cigarette smoke did not interfere with the separation and preparation of 5-HMF. Conclusion: This study realized the simple, rapid, efficient and stable separation and preparation of 5-HMF in cigarette smoke, which not only provided material and technical guarantee for the subsequent systematic study of the toxic effects of important components of cigarette smoke, but also provided methods and ideas for the separation and preparation of other components in the complex matrix of cigarette smoke.