The invention provides an automatic focusing device for an imaging flow cytometer, and belongs to the field of optical instruments in biology and medicine. The device comprises a focusing detector, a focusing lens, a light splitting plate, a laser, a microscope objective, a position sensor and a micro displacement mechanism, wherein the laser is used for emitting collimation laser with field angles, the collimation laser is reflected by the light splitting plate to be focused through the microscope objective on cells to be measured in a liquid flow chamber, the back scattered light of the cells to be measured passes through the microscope objective to penetrate through the light splitting plate and is converged into the position sensor through the focusing lens, the position sensor is used for calculating the defocusing amount, and the defocusing amount is fed back to the position sensor and the micro displacement mechanism which are arranged on the microscope objective; the focusing function is realized through the movement of the micro displacement mechanism and the measurement of the position sensor. The device adopts a laser spot imaging method, and the defocusing amount of the cells can be directly and accurately obtained; the defocusing amount of the cells is tested through the back scattering of the cells, the usage ratio of the energy is higher, and the signal to noise ratio of the whole system is better; the volume of the system is more compact and simpler.