Objective Two sample pretreatment method for the effective extraction and purification of rhodamine B in chili oil were investigated in detail. Valuable information for the accurate assay of rhodamine B by ultra-high performance liquid chromatography with fluorescence detection (UPLC-FLR) was provided. Methods The chili oil was extracted with acidified acetonitrile and then purified by a mixed type cation exchange solid phase extraction (SPE) column in the first method. In the second method, n-hexane containing 20% acetone was used to extract rhodamine B. The extraction was then purified by a neutral alumina SPE column. Both of the purified sample solution was blown to nearly dryness, re-dissolved in 50% methanol-water, filtrated and then analyzed by UPLC-FLR. Results The recoveries at three spiked levels (5.0,0.0,0.0 μg/kg, respectively) were in the ranges of 82.8%-101.9% and 80.8%-93.6%, with relative standard deviations ranging from 1.5%-2.9% (n=6) and 1.0%-2.1% (n=6), respectively. Conclusion Both of the two pretreatment method could meet the requirements of accurate determination of rhodamine B in chili oil. However, the recoveries at spiked levels (5.0 and 50.0 μg/kg) of the first method were higher than those of the second method. Furthermore, no large capacity SPE column was needed. There was no significant difference between recoveries of the two method at high spiked level (200.0 μg/kg).