The invention discloses a single molecule positioning method based on high-speed phase-locked detection, which can perform high-precision single molecule positioning and imaging on a fluorescent sample under the condition of using a single
laser light source. Molecular excitation of a sub-
diffraction limit region is generated by using nonlinear characteristics of excitation and
fluorescence emission when a
fluorescence sample is excited by
laser, and meanwhile, positioning information is obtained by surrounding a single molecule of a
region of interest by using an illumination
light beam; and the
laser intensity is continuously improved, and the positioning precision is also improved until sub-five-nanometer precision information is obtained. And the detection end adopts a high-speed lock-in
amplifier to lock
fluorescence signal information of characteristic frequency in real time, and meanwhile, the final
signal-to-background ratio is also improved. Compared with traditional high-precision positioning based on double
laser light sources, the method has the advantages of a single
laser light source, does not need special fluorescent dye, does not need special matching between double-
wavelength lasers, and has universality; and the damage caused by
phototoxicity and
photobleaching due to over-high illumination intensity is avoided.