In this study, a novel method was proposed to sensitively determine aflatoxin B1 (AFB1) in peanut sample by using a carbon quantum dots-coated dummy molecularly imprinted polymer (CDs-DMIP) monolithic column for pretreatment coupled with high performance liquid chromatography-fluorescence detection (HPLC-FLD). The CDs-DMIP monolithic column was prepared by in-situ polymerization in a water bath using 5,7-dimethoxycoumarin as dummy template molecule. The CDs-DMIP monolithic column was applied to determine AFB1 by HPLC-FLD. Satisfactory linearity was obtained over 0.5–2000 ng mL−1, with a high correlation coefficient of 0.9999. The recoveries of AFB1 in peanut sample ranged from 79.5% to 91.2%, and the intraday and interday relative standard deviation ranged from 1.2% to 4.9%. Limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were 0.118 ng mL−1 and 0.393 ng mL−1, respectively. Under the optimized conditions, the enrichment factor was over 71-fold. AFB1 in peanut sample and even some other samples could be sensitively determined by CDs-DMIP-HPLC-FLD method.