The invention belongs to the technical field of microbiological detection methods, and particularly relates to a microbiological separation and detection method capable of improving the flow range control of a subsequently dropwise added culture solution. The method comprises the following steps: 1, diluting a microbial suspension, and mixing a small amount of diluent with an agar culture medium; 2, uniformly shaking the mixture to prepare a bacteria-containing agar plate; step 3, dropwise adding a culture solution to the local part of the bacterium-containing agar plate at intervals through liquid dropping culture equipment to form dispersed single bacterial colonies, shaking the base station fixing plate in a small range through a shaking assembly, and diffusing the range of the culture solution; 4, selecting bacterial colonies, and performing purification culture; the liquid dropping culture equipment comprises a culture box, cover plates, a frame opening, a light-transmitting plate, sealing frames, rubber strips, a material conveying pipe and a base station fixing plate, the cover plates are installed on the two sides of the culture box, the frame opening is formed in the top of the culture box, the light-transmitting plate is installed on the frame opening, and the sealing frames are symmetrically connected to the two sides of an opening in the upper side of the light-transmitting plate in a sliding mode.