The invention discloses a method for utilizing fluorescence spectrometry for measuring generation of vesicae. The method comprises the steps of taking colorimetric tubes, adding surfactants into the tubes respectively, then adding p-methyaniline solutions with different volume gradients into the tubes respectively, putting the tubes into an ultrasonic apparatus, dropwise adding a tetrahydrofuran solution of Nile red while ultrasonic treatment is conducted, then adding redistilled water for a volume fixing, shaking the mixture uniformly, putting the tubes into the ultrasonic apparatus for 15 min of ultrasonic treatment, conducting standing on a volume fixing mixed system for 4 h under the room-temperature environment then vibrating and shaking uniformly the system, pouring the mixed systeminto a cuvette, and then conducting fluorescence measurement under the same testing condition, wherein if the maximal value of the fluorescence intensity of the mixed system is firstly increased and then decreased along with the increase of the addition amount of p-methyaniline, it is shown that the vesicae are generated, and if the maximal value of the fluorescence intensity of the mixed system is continuously lowered, it is shown that no vesicae are generated. The measuring method is easy to operate and low in cost, the situation that the vesicae are broken when a common transmission electron microscope is vacuumized is avoided, and the situation that high cost is caused when a cryo-electron microscope is adopted for scanning is also avoided.