Detection limit is defined as the minimum concentration or content of a substance which can produce a reliable analytical signal, including the method detection limit and the instrumental detection limit. The instrumental detection limit relies only on the analysis ability of instrument, and the method detection limit is a collection of the pretreatment and detection ability of instrument. Detection limit, as one of the most important parameters of method to evaluate the detection ability, can be analyzed by different calculation methods, including 3N/S, IUPAC, HPLC, EPA, Hubaux-Vos and mathematical model. However, the difference among these methods and their application were not definite. In this study, the detection limit of citrinin using ultra-high performance liquid chromatography fluorescence detector was calculated by different methods. The results showed that the instrumental detection limit of citrinin was in the range of 0.009~0.344 ng/mL. The mathematical model was reliable due to the considered variation of standard deviation. In conclusion, the comparison of characteristics of calculation methods in chromatography can provide a reference for practical application of detection limit in detection process.