The present invention discloses a fluorescence spectrum detection method of soil aryl sulfatase enzymatic activity, and relates to the field of methods for detecting enzymatic activity in soil. The method comprises the following steps: 1, reagent preparation, wherein reagents comprise an aryl sulfatase enzyme fluorescence substrate, an acetate buffer solution, a sodium hydroxide terminating agent,an MU standard substance and a sodium azide solution; and 2, an experimental process, wherein the experimental process comprises (1) microwell plate preparation; (2) soil sample preparation; and (3)a detection process: using an 8-channel pipette to absorb and move soil suspension liquid separately to all columns of a sample group, then placing a microwell plate in an incubator with a standard temperature of 30 degrees for three hours; taking out the microwell plate, adding 50 muL of 0.5 M NaOH to all holes to terminate detection; setting parameters of a microplate reader, then placing the microwell plate into a feeding hole, and reading a measured value on a microplate reader fluorescent screen; and finally performing calculation. The method is simple in operation. Compared with originaldetection methods, an analysis result is stable and reliable, reproducibility is good, and a medicine dosage is small.