The invention relates to a preparation method and an application of a block polyether-beta-CD comb type demulsifying agent for demulsifying and dewatering crude oil. The invention can improve the demulsifying and dewatering efficiency of a crude oil demulsifying agent and the molecular weight and the compounding capacity of the demulsifying agent. The preparation method comprises the following steps of: dissolving beta-cyclodextrin in dimethysulfoxide (DMSO), using NaOH as a catalyst and slowly dropping allylbromide to react for 48 hours at a temperature of 0-10 DEG C to obtain allyl-beta-cyclodextrin; dissolving the allyl-beta-cyclodextrin and acrylic acid raw materials in water, and under the protection of nitrogen, carrying out a polymerization reaction for 8 hours by using NaHSO3 and (NH4)2S2O7 as an initiating system so as to obtain a cyclodextrin/acrylic acid main chain polymer; and finally, putting the main chain polymer and polyoxy-ethylene block polyether in a reactor to react for 12-16 hours to obtain a product, and extracting and centrifugally separating the product to obtain the block polyether-beta-CD comb type demulsifying agent. The preparation method has simple operation, moderate condition, simple equipment and easy industrial production. After the block polyether-beta-CD comb type demulsifying agent is mixed with crude oil, the water yield of the crude oil ishigh, and the demulsifying time is short, thus the block polyether-beta-CD comb type demulsifying agent can be widely applied to oil fields and refineries for demulsifying and dewatering.