The invention relates to a bead-like nanofiber fuel filter membrane and a preparation method thereof. The preparation method comprises the following steps of firstly, adding a non-solvent into a polymer solution to obtain a microgel polymer solution; then putting an electrospun fiber membrane into the microgel polymer solution for dipping, taking out the electrospun fiber membrane, putting the electrospun fiber membrane into an ultrasonic-assisted non-solvent coagulating bath, and increasing Rayleigh instability in a polymer impregnation liquid phase separation process by utilizing ultrasonic waves, thereby preparing a bead-like nanofiber membrane; then making the bead-like nanofiber membrane subjected to hydrophobic treatment; and finally preparing the bead-like nanofiber fuel filter membrane. In the prepared bead-like nanofiber fuel filter membrane, the average particle size of beads is 20-1000 nm, the hydrophobic angle of the filter membrane is 110-160 degrees, the rolling angle is less than or equal to 10 degrees, the average pore size is 200-1000 nm, and the porosity is 80-95%. The method is simple and easy to implement, the roughness of the surface and the pore channel wall of the prepared filter membrane is remarkably increased, the pore diameter is reduced, meanwhile, high porosity is still kept, and high-precision and high-flux filtration of submicron emulsified water in fuel is facilitated.