The invention relates to a preparation method of a gasoline desulfurization microsphere catalyst. The method is characterized by: adding activated zinc oxide accounting for 30-60wt% of the total dry basis of the catalyst, expanded perlite powder and/or diatomite accounting for 5-20wt% of total dry basis, and a nickel salt, in terms of nickel oxide, accounting for 10-30wt% of the total dry basis into chemical water for mixing and beating, and raising the temperature to 60-90DEG C, at the same time continuously adding a sodium aluminate solution and an aluminum sulfate solution with a concentration of 1-20 wt%, adjusting and controlling the flow ratio of the two solutions to maintain the pH of the whole slurry at 7.5-9.5; and with the total amount of alumina in the sodium aluminate and aluminum sulfate accounting for 5-20wt% of the total dry basis of the catalyst, atomizing the uniformly mixed material slurry into microspheres with an average particle diameter of 50-80 microns, then conducting washing with chemical water till sodium oxide is less than 1.0wt%, and after drying, carrying out roasting at a temperature of 450-650DEG C for 0.5-4 hours. The prepared catalyst has a pore volume of 0.1-0.4 ml/g, and is characterized by good strength, high activity, large sulfur capacity, as well as low loss of liquid yield and gasoline octane number in a gasoline desulfurization reaction.