The invention provides an N-trimethyl chitosan microsphere, and a preparation method and an application thereof in reducing dielectric loss of mineral oil, and belongs to the field of modified chitosan. The preparation method comprises the following steps: mixing chitosan, sodium iodide, and N-methyl-2-pyrrolidone to carry out reactions, then adding sodium hydroxide and methyl iodide to carry outreactions; adding ethanol to carry out precipitation, performing separation and freeze-drying to obtain N-trimethyl chitosan with a quaternization substitution degree of 10-20%; preparing a N-trimethyl chitosan solution, spraying the N-trimethyl chitosan solution into sodium polyphosphate to carry out crosslinking reactions; washing the reaction product by water, filtering, adding the reaction product into a sodium hydroxide solution, stirring, adding epoxy chloropropane to carry out reactions to obtain microspheres; washing the microspheres by water, then washing microspheres by organic solvents with volume percentages of 10-20%, 30-40%, 70-80%, and 90-95%, and carrying out freeze-drying. The prepared modified chitosan can reduce the dielectric loss of mineral oil.