In order to clarify the flavonoids compositions of rape bee pollen and evaluate the assisted extraction effect for pulsed electric field (PEF), the ethanol extract of rape bee pollen and its acidolysis component were analyzed in detail by the method of TLC—DPPH and HPLC in this paper. The results showed that the quercetin and kaempferol in the ethanol extract of rape bee pollen could not be detected for their low contents. However, a large number of quercetin and kaempferol were generated after the acidolysis of ethanol extract, and this implied that the flavonoids compositions of rape bee pollen mainly existed in the form of flavonoid glycoside. In addition, the content of quercetin and kaempferol in the acidolysis of ethanol extract were 764.39 μg/g and 1 647.97 μg/g respectively when the acidolysis materials were not treated by PEF. Nevertheless, when the acidolysis of ethanol extract was pretreated by the PEF afterwards, the content of quercetin and kaempferol reached 867.93 μg/g and 1 924.03 μg/g, increasing by 13.55% and 16.75% respectively. These results fully proved that the PEF technology pretreatment was an effective assisted extraction method and could be conducive to break the walls for the rape bee pollen cells.