The inhibitory effects of Portulaca oleracea L. (PO) and its flavonoid ingredients on the formation of heterocyclic amines (HAs) in roast beef patties were investigated. Ten HAs were found in control patties, and the total content was 212.73 ± 7.13 ng/g. With the addition of PO (1%, 5%, and 10%, w/w), HAs decreased by 62.39%, 68.03%, and 73.75%, respectively. The main flavonoid ingredients (rutin, hesperidin, and flavanone) also present a similar inhibitory effect. The Density Function Theory (DFT) methods were adopted to investigate the inhibitory mechanism. These ingredients bonded with the intermediate to block the formation of norharman. Both experimental and calculated data of the ingredients were analyzed on their HAs inhibitory capacity. Our results provide a novel and valuable strategy to reduce HAs via a low additive level of medicinal and edible plants. And the correlation between experimental and calculated data could be applied to predict the inhibitory ability of inhibitors.