A fluorescence microscopy imaging system is used for detecting a fluorescence signal of a sample, and includes a module for detecting fluorescence and a module for focusing control. The module for detecting fluorescence includes a fluorescence excitation light source generator (FELSG) and a fluorescence detector. The FELSG is capable of generating an excitation light beam having a first wavelength to excite the sample to emit fluorescence. The fluorescence detector is used to read the fluorescence signal of the sample. The module for focusing control generates a servo light beam having a second wavelength. A servo light beam reflecting film disposed on an observation plane is used to reflect the servo light beam. A return beam signal is analyzed using a focusing detection method. An actuator is used to move the objective for focusing, so as to enable the fluorescence excitation light beam to excite the sample to emit fluorescence.