A high-efficiency actinomycete separation culture device comprises a support, a plurality of vertical rails on the right side of the support, sliders on the vertical rails, a circular ring at the upper end of the sliders, a dilution tube on the inner ring of the circular ring, a dropper at the bottom of the dilution tube, a first one-way pressure valve in the dropper, a first piston on the dropper, a second through hole on the first piston, and a first electromagnetic valve connected with the second through hole. The first electromagnetic valve is connected with a sterile water supply pipe, the slider is connected with a driving device, and an electric push rod is arranged at the upper end of the support. A controller is also included. In use, the invention is controlled by the controller, sterile water in all the dilution pipes can be automatically supplemented, the dilution ratio of actinomycetes in all the dilution pipes can be ensured to be constant, and the problem of overlarge error caused by single dilution is avoided through a sequential dilution method, so that the device is applicable to supply of a plurality of culture medium plates; especially, the assembly line of a production workshop is adapted, and supplement of solutions of the dilution tubes on different layers and liquid outlet of the dropper on the lowermost side do not conflict with each other.